Methods and compositions related to induced sensory neurons

ABSTRACT

This invention provides methods of generating induced sensory neurons (iSNs) from non-neuronal cells such as fibroblasts. The invention also provides methods of using iSNs in various therapeutic or non-therapeutic applications, e.g., methods to identify agents or cellular modulations that enhance iSN formation from non-neuronal cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The subject patent application is a divisional of U.S. patentapplication Ser. No. 15/106,460 (filed Jun. 20, 2016; now pending),which is a § 371 U.S. national phase filing of PCT International PatentApplication No. PCT/2014/071528 (filed Dec. 19, 2014; now expired),which claims the benefit of priority to U.S. Provisional PatentApplication No. 61/918,965 (filed Dec. 20, 2013; now expired). The fulldisclosures of the priority applications are incorporated herein byreference in their entirety and for all purposes.

BACKGROUND OF THE INVENTION

Sensory neurons of the dorsal root ganglia (DRGs) and trigeminal nervecan detect environmental changes through projections in the skin. Theseneurons comprise three main classes devoted tonociception/puritoception, mechanosensation and proprioception.Nociception is the process by which noxious stimuli such as heat andtouch cause the sensory neurons (nociceptors) in the skin to sendsignals to the central nervous system. Puritoception is the perceptionof itch. Mechanosensation is the detection of pressure, andproprioception is the detection of muscle movement through themonitoring of muscle stretch. The control of pain, itch and disordersthat affect various types of sensory neurons has long been a majorchallenge for pharmacotherapy. It was reported that at least 116 millionAmericans and 35% of the population worldwide suffer from chronicneuropathy or pain (Elzahaf et al., Curr. Med. Res. Opin. 28, 1221-1229,2012). However, only 30% of patients in chronic pain respond to “goldstandard” FDA approved treatments (Finnerup et al., Pain 150, 573-581,2010). Laboratory studies have demonstrated a large range ofinter-individual variation in response to identical pain stimuli.Furthermore, it is becoming increasingly recognized that genetic factorsare a major contributor to pain phenotype and response topharmacological treatments. Despite these findings, much pain relateddrug discovery has relied on animal models, which are unlikely to beuseful in modeling subtle differences between humans and have lowerthroughput than in vitro screens. These factors may help to explain thestaggeringly high rates of attrition during clinical trials for evenpromising preclinical candidates. The control and detection of itch ispoorly understood and also a significant unmet medical need. Althoughcadaveric human sensory neurons are available for research, these cellscannot be genetically altered and have limited availability.

There is a need in the art for effective means for generatingfunctionally responsive sensory neurons in vitro in sufficient numbersfor mechanistic studies or drug screening. The present invention isdirected to this and other unmet needs in the art.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for generatinginduced sensory neurons (iSNs). The methods entail co-expressing in anon-neuronal cell a combination of Brn3A/Ngn1 (BN1) genes or Brn3A/Ngn2(BN2) genes. Typically, expression of the Brn3A/Ngn1 genes or Brn3A/Ngn2genes in the non-neuronal cell is temporal. In some methods, theBrn3A/Ngn1 genes or Brn3A/Ngn2 genes are transiently expressed in thenon-neuronal cell. For example, the Brn3A/Ngn1 genes or Brn3A/Ngn2 genescan be expressed in the cell via an inducible expression system such asinducible expression vectors. In some methods, the non-neuronal cell forconversion into a sensory neuron is a fibroblast, an embryonic stem cell(ESC), or an induced pluripotent stem cell (iPSC). In some methods, thenon-neuronal cell is an embryonic fibroblast or an adult fibroblast.Some methods of the invention are directed to inducing sensory neuronformation from mammalian fibroblast, e.g., fibroblast derived fromhuman, mouse or rat. In some methods, the Brn3A gene and the Ngn1 geneare co-expressed in the non-neuronal cell. In some other methods, theBrn3A gene and the Ngn2 gene are co-expressed in the non-neuronal cell.In some methods, an expression vector harboring the Brn3A gene and theNgn1 or Ngn2 gene is introduced into the non-neuronal cell. For example,a lentiviral vector expressing the BN1 or BN2 genes can be used forinducing the non-neuronal cell. Some methods of the inventionadditionally involve examining the induced sensory neurons for thepresence of one or more neuronal markers.

In some related embodiments, the invention provides induced sensoryneurons generated in accordance with the methods described herein. Insome other embodiments, the invention provides isolated cells thatharbor one or more expression vectors that express a combination ofBrn3A/Ngn1 genes or Brn3A/Ngn2 genes. Some of these cells arenon-neuronal cells. For example, the cell can be a fibroblast, anembryonic stem cell (ESC), or an induced pluripotent stem cell (iPSC).In some embodiments, the cell is an embryonic fibroblast or an adultfibroblast. In some preferred embodiments, the fibroblast is derivedfrom a mammal, e.g., human, mouse or rat. In some of the cells, theexpression vectors co-expressing the Brn3A/Ngn1 genes or Brn3A/Ngn2genes are lentiviral vectors. In some embodiments, the expressionvectors are inducible vectors.

In another aspect, the invention provides methods for treating in asubject a neurological condition or disorder that is associated with ormediated by a loss or degeneration of sensory neurons. The methodsentail (1) obtaining a population of non-neuronal cells from the subjectin need of treatment; (2) generating a population of induced sensoryneurons (iSNs) from the non-neuronal cells by co-expressing in thenon-neuronal cell a combination of Brn3A/Ngn1 genes or Brn3A/Ngn2 genes;and (3) administering a therapeutically effective amount of the iSNpopulation to the subject. Typically, the non-neuronal cells transientlyexpress the Brn3A/Ngn1 genes or Brn3A/Ngn2 genes. In some methods,expression of the Brn3A/Ngn1 genes or Brn3A/Ngn2 genes in thenon-neuronal cell is temporal. In some embodiments, the non-neuronalcells are fibroblast, embryonic stem cells (ESCs), or inducedpluripotent stem cell (iPSCs). In some preferred embodiments, thesubject is human, and the non-neuronal cells are human fibroblast. Insome methods, the Brn3A/Ngn1 genes or Brn3A/Ngn2 genes are introducedinto the non-neuronal cells via a lentiviral vector.

In another aspect, the invention provides methods for identifying anagent or cellular modulation that stimulates conversion of anon-neuronal cell into an induced sensory neuron (iSN). These methodsentail (1) co-expressing in a non-neuronal cell (1) a Brn3A gene and (2)a Ngn1 or Ngn2 gene in the presence of candidate compounds or cellularmanipulations, and (2) detecting enhanced iSN conversion from thenon-neuronal cell that has been subject to contact with a specificcandidate compound (or subject to a specific cellular manipulation)relative to iSN conversion from the non-neuronal cell that has not beensubject to contact with the specific candidate compound (or subject tothe specific cellular manipulation). The methods allow identification ofthe specific candidate compound (or cellular manipulation) as an agentthat stimulates conversion of a non-neuronal cell into an inducedsensory neuron. Typically, expressions of the Brn3A/Ngn1 genes orBrn3A/Ngn2 genes in the non-neuronal cells are transient. In somepreferred embodiments, expression of the Brn3A/Ngn1 genes or Brn3A/Ngn2genes is temporally controlled (e.g., via using inducible expressionvectors). In some methods, the candidate compounds to be screened aretranscription factors or miRNAs. Some other methods are directed toscreening cellular manipulations such as epigenetic modulations of thenon-neuronal cell. In some methods, the non-neuronal cell used in thescreening is a fibroblast, an embryonic stem cell (ESC), or an inducedpluripotent stem cell (iPSC). For example, some screening methods of theinvention employ an embryonic fibroblast or an adult fibroblast, e.g., amammalian fibroblast derived from human, mouse or rat. In some preferredembodiments, the Brn3A/Ngn1 genes or Brn3A/Ngn2 genes are introducedinto the non-neuronal cell via a lentiviral vector.

A further understanding of the nature and advantages of the presentinvention may be realized by reference to the remaining portions of thespecification and claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that transient co-expression of two developmentallyrelevant transcription factors stably reprograms fibroblasts to attainproperties of functionally mature neurons. (a) Transient co-expressionof Brn3a with either Ngn1 or Ngn2 for 8 days in fibroblasts inducescells with neural morphology that stain for pan-neural markers Map2 andTuj1 Cells were immunostained 14 days post-induction. Scale bar: 100 μm.(b) Cooperative expression of Brn3a with Ngn1 or 2 is required forinduction of Map2/Tuj1 double positive cells. TFs were induced for 8days and immunostained 14 days post-induction. Bars and error representmeans and SEMs from three replicates. (c) Synapsin expression in BN2neural cells, cells were counterstained for pan-neural marker Tuj1. (d)Vamp expression in BN1 neural cells. Scale bars: 25 μm (e) The majorityof neurons induced with BN1 and BN2 express synaptic marker indicativeof mature neurons. Bars in graphs represent means and error barsrepresent ±SEM. (f) Whole-cell currents recorded in voltage-clamp mode.Inward fast inactivating Na⁺ and outward currents are observed. (g)Representative action potential evoked from MEFs after 14 days inculture. (h) A train of spontaneous action potentials observed in 14-dayculture.

FIG. 2 shows that neurons induced with BN1 or BN2 exhibit molecularhallmarks of the somatic sensory neural lineage. (a) Quantification ofneurofilaments, neurotransmitters, and neuropeptide expression in BN1and BN2 neurons. Expression was assessed 20 days post induction viaimmunostaining. Bars represent mean, error bars represent SEM from atleast 100 cells. (b) A subset of neurons induced via BN1 or 2 expressthe peripheral neurofilament peripherin. Scale bar: 25 μm. (c) A subsetof neurons induced via BN1 or 2 express the peripheral neurofilamentNF200. Scale bar: 25 μm. (d) Time course of quantitative RT-PCR analysesof Isl1 and endogenous Brn3a expression following induction ofBrn3a/Ngn1 or Brn3a/Ngn2. Fold-induction calculated as the increase inexpression from un-induced fibroblasts cultured for the same duration inthe same conditions for each time-point. Doxycycline was withdrawnpermanently eight days post-induction as indicated with the arrow. (e)Single cell quantitative RT-PCR for of induced neurons 20 dayspost-induction. (f) Fold-induction of TrkA, B, and C following inductionof Brn3a/Ngn1 or Brn3a/Ngn3. Fold-induction is calculated as theincrease in expression from un-induced fibroblasts cultured for the sameduration in the same conditions for each time-point. (g) Quantificationof neurons positive for p75 and each of the three Trk receptorsindividually and combined. Bars represent means and error bars representSEM from two independent experiments in which a minimum of 150 cellswere counted. (h) Representative immunostaining for Trk A, B, and C 20days post-induction. (i) p75 immunostaining 20 days post induction.Scale bar: 25 μm.

FIG. 3 shows that reprogramming induces somatic sensory neuralmorphology. (a) TrkA, TrkB, and TrkC immunoreactive neurons havedistinct distribution of soma size. Graph depicts mean soma areas by Trkimmune-reactivity. Error bars represent ±SEM. ***p<0.001;**p<0.01(one-way ANOVA with Newman-Keuls post-hoc comparison) (b)Typical morphology of pseudounipolar cells induced by BN1 and BN2. Scalebar: 100 μm (c) The majority of Map2/Tuj1-positive cells induced viaBrn3a/Ngn1 or Brn3a/Ngn2 are pseudo-unipolar. The figure showsquantification of the neural morphologies observed in representativeexperiments fourteen days post-induction. Bars represent means from twoindependent experiments.

FIG. 4s show that iSNs possess functional properties of sensory neurons.(a) RT-PCR analysis of MEFs, neurons induced with BAZ and iSNs generatedwith BN1 or BN2. TrpA1, TrpM8, TrpV1 and Nav1.7 are detected in BN1 andBN2 but not in MEFs or BAZ. (b) Quantitative RT-PCR of MEFs, neuronsinduced with BAZ and iSNs generated with BN1 or BN2. BAZ, BN1 and BN2samples expressed similar levels of Map2. TrpA1, TrpM8, TrpV1 and Nav1.7are present in BN1 and BN2 however not detected in MEFs or BAZ samplesindicated by n.d. Expression is normalized to Gapdh. Expression levelsare relative to BN1 such that expression of BN1=1.0. Bars and error barsrepresent means and SEMs from two independent biological replicates. (c)Representative calcium responses for 10 μM capsaicin (Cap), 100 μMMenthol (Menth), and 100 μM mustard oil (MO). Calcium transients weremeasured using Map2::GCAMP5.G. Calcium responses were calculated as thechange in fluorescence (ΔF) over the initial fluorescence (F_(o)).Depolarization with 2.5 mM KCl was used at the beginning and end of eachexperiment to confirm neural identity and sustained functional capacity.(d) ΔF/F_(o) intensity plot showing the response of individual cells toeach ligand. Each cell is represented in each column. Cells respond toeither KCl only (black circle), KCl plus one other ligand (othercircles), or KCl plus two other ligands (diamond, triangle or square).(e) Distribution of KCl responders that responded to either KCl only,KCl plus one other ligand, or KCl with two other ligands. Bars representmeans from at least four experiments. Error bars represent SEM.

FIG. 5 shows that induction of the somatic sensory lineage does notrequire cell division or specialized embryonic cells. (a and b) EdU andMap2 staining fourteen days post induction. (c) Quantification of thenumber of Map2-positive cells that co-stained for the mitotic indicatorEdU. rtTA are MEFs infected with reverse tetracycline trans-activator.BAZ is MEFs infected with Brn2, Mash1 (also known as Ascl1) and Zic1 apreviously reported transcriptional cocktail for the direct conversionof MEFs to neurons¹⁰. Bar are means from two separate experiments. ErrorBars are SEM. Scale bar: 25 μm (d and e) BN1 and BN2 generate neurons inthe presence of the mitotic inhibitor AraC. AraC was applied from threedays post induction until the end of the experiment at 4 μM, aconcentration empirically determined to inhibit >90% proliferativecells. (e) Mitotic inhibition does not significantly decrease the numberof neurons generated by BN1 or BN2. Bars are means of two replicateexperiments. Error bars are SEM. (g) Quantification of Map2/Tuj1positive cells induced from tail-tip fibroblasts. (h) BN1 and BN2 induceneural cells from TTFs that stain for somatic sensory markers. Scalebar: 25 μm.

FIG. 6 shows that human iSNs are generated using BN1 and BN2. (a)Expression of BN1 and BN2 converts human embryonic fibroblasts toMAP2/TUJ1 double positive cells with neuronal morphologies 14 days afterinduction, dox was removed on day 8. Scale bar: 100 μm (b) percent TUJ1single positive (grey) and MAP2/TUJ1 double positive cells generatedfrom HEFs in BN1, BN2, and rtTA control conditions. (c-1) Neuronsinduced with BN1/2 express somatic sensory markers Scale bar: 25 □□ m.(m) Quantitative RT-PCR of TRK receptors in MEFs and iSNs generated withBN1 or BN2. Expression levels are normalized to MAP2. Bars and errorbars represent means and SEMs from two independent biologicalreplicates. (n) Quantification of TUJ positive cells expressing TRKA, B,C, VGLUT2 and ISL1 in BN1, BN2, BAZ and rtTA control conditions. (o)Quantification of TUJ positive cells expressing NF200, PRPH, and RET inBN1 and BN2. Error bars represent SEM.

FIG. 7 shows that human iSNs possess functional properties of sensoryneurons (a,b) Representative traces of Na/K currents and evoked actionpotential in BN1 and BN2 iSNs. (c) PCR analysis showing presence ofligand gated calcium channels TRPA1, TRPM8, TRPV1, and voltage gatedsodium channel Nav1.7 in BN1 and BN2 human iSNs compared to BAZ anduninfected controls (HEFs) at 16days post induction. BN1, BN2, and BAZsamples express MAP2 compared to uninfected controls. TRPA1, M8, V1, andNav1.7 are present only in BN1 and BN2. GAPDH amplification in samplesprovides loading control, and GAPDH used in the absence reversetransciptase (-RT) controls for contaminating genomic DNA. (d)Representative calcium responses for 10 μM capsaicin (Cap), 100 μMMenthol (Men), and 100 μM mustard oil (MO). Calcium transients weremeasured using Map2::GCAMP5.G. Calcium responses were calculated as thechange in fluorescence (ΔF) over the initial fluorescence (F_(o)).Depolarization with 2.5 mM KCl was used at the beginning and end of eachexperiment to confirm neural identity and sustained functional capacity.(e) ΔF/F_(o) intensity plot showing the response of individual cells toeach ligand. Each cell is represented in each column. Cells respond toKCl only (black circle), KCl plus one other ligand (the other circles),or KCl plus two other ligands (triangle or square). (f) Distribution ofKCl responders that responded to either KCl only, KCl plus one otherligand, or KCl with two other ligands. Bars represent means from sevenexperiments. Error bars represent SEM. (g) Representative calciumresponses for 100 μM Histamine (Hist), 100 μM Chloroquine (CQ), 10 μMBAM8-22 (BAM), 10 μM SLI-GRL (SLI). (h) ΔF/F_(o) intensity plots ofthree separate ligand combination regimes showing the response ofindividual cells to each ligand. Each cell is represented in eachcolumn. Cells respond to KCl only (black circle), KCl plus one otherligand (the other circles), or KCl plus two other ligands (triangles orsquares). (i) Distribution of KCl responders in BN2 that responded toeither KCl only, KCl plus one other ligand, or KCl with multipleligands. Bars represent means from at least 2 experiments. Error barsrepresent SEM.

FIG. 8 shows that ectopic expression of BN1 or BN1 mediates neuralreprogramming. (a) Doxycycline inducible lentiviral vectors (b) Neuralinduction methods. (c) Representative staining for single and doubleectopic expression of BN1 and BN1 in MEFs using protocol described inS1a.

FIG. 9 shows that BN1 and BN2 neurons possess molecular signatures ofsensory neurons. (a) iSNs induced with either BN1 or BN2 are excitatoryneurons show immunostaining for vGlut1, vGlut2, and vGlut3, CGRP in Tuj1positive cells. Scale bars represent 25 μM. (b) Brn3a but not Ngn1/2expression persists following dox withdrawal. (c) Quantification ofneurons positive for each of the three Trk receptors individually and inpair wise combination. Bars represent means and error bars represent SEMfrom two independent experiments in which a minimum of 100 cells wascounted.

FIG. 10 shows analysis of iSN morphologies. (a) TrkA, TrkB, and TrkCneurons have distinct distribution of soma size. Scatter plot of somaareas by Trk receptor immune-reactivity. (b) Table present statisticalattributes of soma size distribution. Representative images of neuronsinduced with Brn2, Ascl1 (also called Mash1) and Zic1(BAZ) and BN1 andBN2. Black is Tuj1 staining. Scale bar 100 μm. (c) representativeneurite branching for iSNs generated with Brn2, Ascl1, Zic1 (BAZ) or BN1and BN2. Neurons were immunostained for Tuj1.

DETAILED DESCRIPTION I. Overview

The present invention is predicated in part on the findings by thepresent inventors of transcription factors that convert mouse and humanfibroblasts into sensory neurons. As detailed herein, it was found thattwo lineage-relevant transcription factors, without additional exogenouscues, can induce neurons that phenocopy many defining signatures ofsomatosensory neurons including morphological features, gene expression,and functional properties. The induced sensory neurons (iSNs) in mouseexhibit two key morphological hallmarks of peripheral sensory neurons,pseudounipolar morphology, and lineage-restricted size distributions.Specifically, it was found that neural reprogramming with co-expressionof the transcription factors, Brn3a/Ngn1 (BN1) or Brn3a/Ngn2 (BN2), ishighly selective with the overwhelming majority of induced neuronsassuming one of three major somatosensory neural lineages and express aspecific combinatorial array of markers unique to somatosensory neurons.Also, no differences were observed between iSNs generated via Ngn1 orNgn2. Furthermore, the inventors were able to recapitulate these inducedsensory neurons in human fibroblasts, and these iSNs possess thefunctional ‘gold-standard’ of nociceptive neurons, the ability to senseand selectively respond to noxious and itch inducing stimuli. Thesefindings indicate that the iSNs are functional phenocopies of peripheralsensory neurons, and as a result are useful in the study of sensorybiology, diseases, and drug screening directly in cells derived fromhumans of diverse genetic backgrounds.

While the observed iSNs are divided into three distinct somatosensorylineages that developmentally arise from common precursors, iSNconversion does not require transiting through a proliferative neuralprecursor. The direct conversion offers some clear advantages togenerating in vitro neuronal models through directed differentiationfrom pluri- or multi-potent stem cells. For example, direct iSNconversion is rapid, efficient, and requires minimal manipulation of theculture environment. Furthermore, avoiding proliferative precursors viadirect conversion limits opportunities for epigenetic changes that coulddeprogram pathogenic or phenotypically desired traits.

In accordance with these studies, the invention provides methods forgenerating induced sensory neurons from non-neuronal cells viaco-expressing a combination of the Brn3a/Ngn1 (BN1) genes or theBrn3a/Ngn2 (BN2) genes. The methods establish a novel and eleganttechnique for directly inducing a unique neuronal sub-type fromnon-neuronal cells such as fibroblasts. Also encompassed by theinvention are non-neuronal cells containing expressing vector(s) fortransiently expressing the BN1 or BN2 genes, induced sensory neuronsthus generated, as well as transgenic non-human animals harboring suchnon-neuronal cells or induced sensory neurons. By utilizing a minimalset of transcription factors, the methods provide a foundation forunderstanding how neural induction proceeds and a platform to screen foradditional factors that enable selective induction of specific sensorysub-populations. Thus, the invention additionally provides methods foridentifying other agents or cellular modulations that can improve theefficiency, accuracy or specificity of iSN induction, includingtranscription factors, miRNA, or other genetic and epigeneticmodulations. Further provided in the invention are methods of employingiSNs generated in accordance with the invention in various therapeuticapplications. The following sections provide guidance for making andusing the compositions of the invention, and for carrying out themethods of the invention.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this invention: Academic Press Dictionary of Science and Technology,Morris (Ed.), Academic Press (1^(st) ed., 1992); Oxford Dictionary ofBiochemistry and Molecular Biology, Smith et al. (Eds.), OxfordUniversity Press (revised ed., 2000); Encyclopaedic Dictionary ofChemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionaryof Microbiology and Molecular Biology, Singleton et al. (Eds.), JohnWiley & Sons (3^(rd) ed., 2002); Dictionary of Chemistry, Hunt (Ed.),Routledge (1^(st) ed., 1999); Dictionary of Pharmaceutical Medicine,Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of OrganicChemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd.(2002); and A Dictionary of Biology (Oxford Paperback Reference), Martinand Hine (Eds.), Oxford University Press (4^(th) ed., 2000). Inaddition, the following definitions are provided to assist the reader inthe practice of the invention.

The term “agent” or “test agent” includes any substance, molecule,element, compound, entity, or a combination thereof. It includes, but isnot limited to, e.g., protein, polypeptide, small organic molecule,polysaccharide, polynucleotide, and the like. It can be a naturalproduct, a synthetic compound, or a chemical compound, or a combinationof two or more substances. Unless otherwise specified, the terms“agent”, “substance”, and “compound” are used interchangeably herein.

The term “analog” is used herein to refer to a molecule thatstructurally resembles a reference molecule but which has been modifiedin a targeted and controlled manner, by replacing a specific substituentof the reference molecule with an alternate substituent. Compared to thereference molecule, an analog would be expected, by one skilled in theart, to exhibit the same, similar, or improved utility. Synthesis andscreening of analogs, to identify variants of known compounds havingimproved traits (such as higher binding affinity for a target molecule)is an approach that is well known in pharmaceutical chemistry.

As used herein, “contacting” has its normal meaning and refers tocombining two or more agents (e.g., polypeptides or small moleculecompounds) or combining agents and cells. Contacting can occur in vitro,e.g., combining two or more agents or combining a test agent and a cellor a cell lysate in a test tube or other container. Contacting can alsooccur in a cell or in situ, e.g., contacting two polypeptides in a cellby coexpression in the cell of recombinant polynucleotides encoding thetwo polypeptides, or in a cell lysate.

Stem cells are cells characterized by the ability of self-renewalthrough mitotic cell division and the potential to differentiate into atissue or an organ. Embryonic stem cells (ESCs) are pluripotent stemcells derived from the inner cell mass of a blastocyst, an early-stageembryo. ESCs are pluripotent, that is, they are able to differentiateinto all derivatives of the three primary germ layers: ectoderm,endoderm, and mesoderm. These include each of the more than 220 celltypes in the adult body. Pluripotency distinguishes embryonic stem cellsfrom adult stem cells found in adults; while embryonic stem cells cangenerate all cell types in the body, adult stem cells are multipotentand can produce only a limited number of cell types. Additionally, underdefined conditions, embryonic stem cells are capable of propagatingthemselves indefinitely. This allows embryonic stem cells to be employedas useful tools for both research and regenerative medicine, becausethey can produce limitless numbers of themselves for continued researchor clinical use.

Induced pluripotent stem cells (iPSCs) are a type of pluripotent stemcell artificially derived from a non-pluripotent cell—typically an adultsomatic cell—by inducing a “forced” expression of specific genes.Induced pluripotent stem cells are similar to natural pluripotent stemcells, such as embryonic stem (ES) cells, in many aspects, such as theexpression of certain stem cell genes and proteins, chromatinmethylation patterns, doubling time, embryoid body formation, teratomaformation, viable chimera formation, and potency and differentiability,but the full extent of their relation to natural pluripotent stem cellsis still being assessed. Induced pluripotent cells have been made fromadult stomach, liver, skin cells, blood cells, prostate cells andurinary tract cells.

Sensory neurons are neurons responsible for converting various externalstimuli that arise from the environment of an organism, producingcorresponding internal stimuli. They are activated by sensory input, andsend projections to other elements of the nervous system, ultimatelyconveying sensory information to the brain or spinal cord. Unlikeneurons of the central nervous system, whose inputs come from otherneurons, sensory neurons are activated by physical modalities such asvisible light, sound, heat, physical contact, etc., or by chemicalsignals for example in the case of smell or taste. Peripheral sensoryneurons are composed of three major lineages, nociceptors,mechanoceptors, and proprioceptors, which are demarcated by expressionof TrkA, TrkB and TrkC, respectively. Peripheral sensory neurons arisefrom two waves of development coordinated by co-expression of Neurogenin1 (Ngn1) and Neurogenin 2 (Ngn2).

Induced sensory neurons (iSNs) refer to cells of the neuronal lineage,i.e. mitotic neuronal progenitor cells and post-mitotic neuronalprecursor cells and mature neurons, that arise from a non-neuronal cellby experimental manipulation. iSNs express markers specific for cells ofthe neuronal lineage, e.g. Tau, Tuj1, MAP2, NeuN, and the like, and mayhave characteristics of functional neurons, that is, they may be able tobe depolarized, i.e., propagate an action potential, and they may beable to make and maintain synapses with other neurons.

miRNA (or microRNA) refers to a class of small RNA molecules that arecapable of modulating RNA translation (see, Zeng and Cullen, RNA,9:112-123, 2003; Kidner and Martienssen, Trends Genet, 19:13-6, 2003;Dennis et al., Nature, 420:732, 2002; and Couzinet al., Science298:2296-7, 2002). MicroRNAs (miRNAs) encompass a family of ˜22nucleotide (nt) non-coding RNAs. These RNAs have been identified inorganisms ranging from nematodes to humans. Many miRNAs areevolutionarily conserved widely across phyla, regulating gene expressionby post-transcriptional gene repression. The long primary transcripts(pri-miRNAs) are transcribed by RNA polymerase II; processed by anuclear enzyme Drosha; and released as a ˜60 bp hairpin precursor(pre-miRNAs). Pre-miRNAs are processed by RNase III enzymes, Dicer, to˜22 nt (mature miRNAs) and then incorporated into RISC (RNA-inducedsilencing complex). The complex of miRNAs-RISC binds the 3′ UTR of thetarget mRNAs and conducts translational repression or degradation ofmRNAs.

The term “modulate” with respect to a reference molecule or cellularactivity (e.g., transcription or DNA methylation) refers to inhibitionor activation of a biological activity of the reference molecule orcellular activity. Modulation can be up-regulation (i.e., activation orstimulation) or down-regulation (i.e., inhibition or suppression). Themode of action can be direct, e.g., through binding to the referencemolecule. The modulation can also be indirect, e.g., through binding toand/or modifying another molecule which otherwise modulates thereference molecule.

Enhanced efficiency of conversion refers to an up-regulated ability of aculture of non-neuronal cells to give rise to the induced sensoryneurons when contacted with a compound (or subjected to a genetic orepigenetic modulation) relative to a culture of the same type of cellsthat is not contacted with the compound (or subjected to themodulation). By enhanced, it is meant that the cell cultures have anability to give rise to induced sensory neurons that is greater than theability of a population that is not contacted with the candidate agentor induction agent, e.g., 150%, 200%, 300%, 400%, 600%, 800%, 1000%, or2000% of the ability of the uncontacted (or unmodulated) population. Inother words, the cell cultures produce 1.5-fold or more, 2-fold or more,3-fold or more, 4-fold or more, 6-fold or more, 8-fold or more, 10-foldor more, 20-fold or more, 30-fold or more, 50-fold or more, 100-fold ormore, 200-fold or more the number of induced sensory neurons as theuncontacted (or unmodulated) population.

“Polynucleotide” or “nucleic acid sequence” refers to a polymeric formof nucleotides (polyribonucleotide or polydeoxyribonucleotide). In someinstances a polynucleotide refers to a sequence that is not immediatelycontiguous with either of the coding sequences with which it isimmediately contiguous (one on the 5′ end and one on the 3′ end) in thenaturally occurring genome of the organism from which it is derived. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA) independent of othersequences. Polynucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide.

A polypeptide or protein refers to a polymer in which the monomers areamino acid residues that are joined together through amide bonds. Whenthe amino acids are alpha-amino acids, either the L-optical isomer orthe D-optical isomer can be used, the L-isomers being typical. Apolypeptide or protein fragment can have the same or substantiallyidentical amino acid sequence as the naturally occurring protein. Apolypeptide or peptide having substantially identical sequence meansthat an amino acid sequence is largely, but not entirely, the same, butretains a functional activity of the sequence to which it is related.

Polypeptides may be substantially related due to conservativesubstitutions. A conservative variation denotes the replacement of anamino acid residue by another, biologically similar residue. Examples ofconservative variations include the substitution of one hydrophobicresidue such as isoleucine, valine, leucine or methionine for another,or the substitution of one polar residue for another, such as thesubstitution of arginine for lysine, glutamic for aspartic acids, orglutamine for asparagine, and the like. Other illustrative examples ofconservative substitutions include the changes of: alanine to serine;arginine to lysine; asparagine to glutamine or histidine; aspartate toglutamate; cysteine to serine; glutamine to asparagine; glutamate toaspartate; glycine to proline; histidine to asparagine or glutamine;isoleucine to leucine or valine; leucine to valine or isoleucine; lysineto arginine, glutamine, or glutamate; methionine to leucine orisoleucine; phenylalanine to tyrosine, leucine or methionine; serine tothreonine; threonine to serine; tryptophan to tyrosine; tyrosine totryptophan or phenylalanine; valine to isoleucine to leucine.

The term “subject” includes mammals, especially humans, as well as othernon-human animals, e.g., horse, dogs and cats.

A “substantially identical” nucleic acid or amino acid sequence refersto a polynucleotide or amino acid sequence which comprises a sequencethat has at least 75%, 80% or 90% sequence identity to a referencesequence as measured by one of the well-known programs described herein(e.g., BLAST) using standard parameters. The sequence identity ispreferably at least 95%, more preferably at least 98%, and mostpreferably at least 99%. In some embodiments, the subject sequence is ofabout the same length as compared to the reference sequence, i.e.,consisting of about the same number of contiguous amino acid residues(for polypeptide sequences) or nucleotide residues (for polynucleotidesequences).

Sequence identity can be readily determined with various methods knownin the art. For example, the BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5,N=−4, and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)). Percentage of sequenceidentity is determined by comparing two optimally aligned sequences overa comparison window, wherein the portion of the polynucleotide sequencein the comparison window may comprise additions or deletions (i.e.,gaps) as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

As used herein, “treating” or “ameliorating” includes (i) preventing apathologic condition (e.g., sensory neuronopathy) from occurring (e.g.prophylaxis); (ii) inhibiting the pathologic condition (e.g., sensoryneuronopathy) or arresting its development; and (iii) relieving symptomsassociated with the pathologic condition (e.g., sensory neuronopathy).Thus, “treatment” includes the administration of an isolated (and/orpurified) iSN population of the invention and/or other therapeuticcompositions or agents to prevent or delay the onset of the symptoms,complications, or biochemical indicia of a disease described herein,alleviating or ameliorating the symptoms or arresting or inhibitingfurther development of the disease, condition, or disorder. “Treatment”further refers to any indicia of success in the treatment oramelioration or prevention of the disease, condition, or disorderdescribed herein, including any objective or subjective parameter suchas abatement; remission; diminishing of symptoms or making the diseasecondition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. Detailed procedures for the treatment or amelioration ofthe disorder or symptoms thereof can be based on objective or subjectiveparameters, including the results of an examination by a physician.

A “variant” of a reference molecule (e.g., a Neurogenin 1 or Neurogenin2) is meant to refer to a molecule substantially similar in structureand biological activity to either the entire reference molecule, or to afragment thereof. Thus, provided that two molecules possess a similaractivity, they are considered variants as that term is used herein evenif the composition or secondary, tertiary, or quaternary structure ofone of the molecules is not identical to that found in the other, or ifthe sequence of amino acid residues is not identical.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother polynucleotide segment may be attached so as to bring about thereplication of the attached segment. Vectors capable of directing theexpression of genes encoding for one or more polypeptides are referredto as “expression vectors”.

A retrovirus (e.g., a lentivirus) based vector or retroviral vectormeans that genome of the vector comprises components from the virus as abackbone. The viral particle generated from the vector as a wholecontains essential vector components compatible with the RNA genome,including reverse transcription and integration systems. Usually thesewill include the gag and pol proteins derived from the virus. If thevector is derived from a lentivirus, the viral particles are capable ofinfecting and transducing non-dividing cells. Recombinant retroviralparticles are able to deliver a selected exogenous gene orpolynucleotide sequence such as therapeutically active genes, to thegenome of a target cell.

III. Brn3a and Ngn1/Ngn2 Genes for Inducing Formation of Sensory Neurons

To generate induced sensory neurons (iSNs) from non-neuronal cells,methods of the invention entail co-expressing the Brn3a gene (genomic orcDNA sequence) and the Ngn1 (or Ngn2) gene (genomic or cDNA sequence) inthe non-neuronal cells. Brn3a (also called Pou4f1) encodes thebrain-specific homeobox/POU domain protein 3A (BRN3A), aka “POU-domaintranscription factor” (POU4F1), which regulates the transition fromneurogenesis to mature terminally differentiated state. Neurogenins area family of bHLH transcription factors involved in specifying neuronaldifferentiation. They are essential for the formation of dorsal rootganglia. Neurogenin 1 (Ngn1) acts as a regulator for neuronaldifferentiation by binding to enhancer regulatory elements on genes thatencode transcriptional regulators of neurogenesis. Ngn1 is a proneuralgene because its expression is seen prior to neural lineagedetermination. In order for Ngn1 to bind with high fidelity to genomicDNA, it must dimerize with another bHLH protein. Neurogenin 2 (Ngn2) isa transcription factor involved in both neurogenesis and neuralspecification. This protein binds to enhancer box regulatory elements onthe promoters of many genes related to neurogenesis and neuralspecification. For sufficient DNA binding, Ngn2 must form a dimer withan enhancer protein.

Brn3a gene and Ngn1/Ngn2 genes from various species can be readilyemployed in the practice of the present invention. Genomic and cDNAsequences of these genes are all known in the art. See, e.g., Trieu etal., Development 130, 111-121, 2003; Lanier et al., Dev. Dyn. 238,3065-3079, 2009; Velkey et al., Dev. Dyn. 242, 230-253, 2013; and Ma etal., Genes Dev. 13:1717-28, 1999. For example, Brn3a genomic and/or cDNAsequences from various species have been reported, cloned, andcharacterized in the literature, including human Brn3a gene, mouse Brn3agene, rat Brn3a gene, chicken Brn3a gene, and zebrafish Brn3a gene. Seee.g., He et al., Nature 340, 35-41, 1989; Collum et al., Nucleic AcidsRes. 20, 4919-4925, 1992; Gerrero et al., Proc. Natl. Acad. Sci. U.S.A.90, 10841-10845, 1993; Bhargava et al., Proc. Natl. Acad. Sci. U.S.A.90, 10260-10264, 1993; Turner et al., Neuron 12, 205-218, 1994; Xiang etal., J. Neurosci. 15, 4762-4785, 1995; Smith et al., J. Biol. Chem. 272,1382-1388, 1997; Fedtsova and Turner, Mech. Dev. 105, 129-144, 2001;Thomas et al., Biochem. Biophys. Res. Commun. 318, 1045-1051, 2004;Aizawa et al., Curr Biol. 15, 238-43, 2005; and Trieu et al.,Development 130, 111-121, 2003. Similarly, Ngn1 and Ngn2 cDNA sequencesfrom human, mouse, rat, chicken, xenopus, zebrafish, and many otherspecies have also been cloned and functionally characterized. See, e.g.,Sommer et al., Mol. Cell. Neurosci. 8, 221-241, 1996; McCormick et al.,Mol. Cell. Biol. 16, 5792-5800, 1996; Ma et al., Cell 87, 43-52, 1996;Gradwohl et al., Dev. Biol. 180, 227-241, 1996; Tamimi et al., Genomics40, 355-357, 1997; Fode et al., Neuron 20, 483-494, 1998; Korzh et al.,Dev. Dyn. 213, 92-104, 1998; Ma et al., Genes Dev. 13:1717-28, 1999;Perez et al., Development 126, 1715-1728, 1999; Franklin et al., J.Child Neurol. 16, 849-853, 2001; Simons et al., Dev. Biol. 229, 327-339,2001; Kim et al., Exp. Mol. Med. 34, 469-475, 2002; Klein et al., Dev.Dyn. 225, 384-391, 2002; and Zimin et al., Genome Biol. 10, R42, 2009.The specific genomic or mRNA sequences corresponding to these genes arealso available from, e.g., GenBank. Any of these Brn3a genes andNgn1/Ngn2 genes can be employed in the practice of the presentinvention.

In addition to the various wildtype Brn3a genes (or cDNA sequences) andNgn1/Ngn2 genes (or cDNA sequences) described above, variants orfunctional derivatives of such genes (or cDNA sequences) can also beused in the invention. Thus, methods of the invention can utilize avariant or modified Brn3a sequence and/or Ngn1 (or Ngn2) sequence thatis substantially identical to its wildtype counterpart, e.g.,conservatively modified variants. For example, the substantiallyidentical variants should contain a sequence that is at least 80%, 90%,95% or 99% identical to the wildtype sequence. In some embodiments, thefunctional derivatives are variants produced by non-conservativesubstitutions to the extent that that they substantially retain theactivities of the native proteins. Modification to a polynucleotideencoding a polypeptide of interest can be performed with standardtechniques routinely practiced in the art. In some other embodiments,the functional derivatives can contain a partial sequence of thewildtype Brn3a gene and/or the Ngn1 (or Ngn2) gene. Such partialsequence should encode a functionally fragment that possesses some orall of the cellular functions of the wildtype protein, e.g., activitiesin regulating neurogenesis. Cellular functions (e.g., transcriptionalregulation) of the BRN3A, Ngn1 and Ngn2 transcription factors have beendelineated in the art. Based on their structural and functionalinformation known in the art, cloning and expression of functionalfragments of these transcription factors can be readily carried out viastandard techniques of molecular biology. In addition, the functionalderivatives of BRN3A, Ngn1 and Ngn2 described herein can be subject tothe screening methods described below to confirm their activities inpromoting formation of induced sensory neurons.

IV. Non-Neuronal Cells for Generating Induced Sensory Neurons

Various non-neuronal cells can be employed in the present invention forgenerating induced sensory neurons. These include fibroblasts, stemcells, blood cells, and other non-neuronal somatic cells. Thenon-neuronal cells can be obtained from both human and non-human animalsincluding vertebrates and mammals. Thus, other than human cells andmouse cells as exemplified herein, the cells can also be from otheranimal species such as bovine, ovine, porcine, canine, feline, avian,bony and cartilaginous fish, rats, other primates including monkeys, aswell as other animals such as ferrets, sheep, rabbits and guinea pigs.

In general, non-neuronal cells suitable for the methods of the inventioncan be any somatic cells that would not give rise to a sensory neuron inthe absence of experimental manipulation. Examples of such non-neuronalsomatic cells include differentiating or differentiated cells fromectodermal (e.g., keratinocytes), mesodermal (e.g., fibroblast),endodermal (e.g., pancreatic cells), or neural crest lineages (e.g.,melanocytes). The somatic cells may be, for example, pancreatic betacells, glial cells (e.g., oligodendrocytes, astrocytes), hepatocytes,hepatic stem cells, cardiomyocytes, skeletal muscle cells, smooth musclecells, hematopoietic cells, osteoclasts, osteoblasts, pericytes,vascular endothelial cells, schwann cells, dermal fibroblasts, and thelike. They may be terminally differentiated cells, or they may becapable of giving rise to cells of a specific, non-neuronal lineage,e.g., cardiac stem cells, hepatic stem cells, and the like. The somaticcells are readily identifiable as non-neuronal by the absence ofneuronal-specific markers that are well-known in the art, as describedherein. Of interest are cells that are vertebrate cells, e.g., mammaliancells, such as human cells, including adult human cells.

Some preferred embodiments of the invention utilize fibroblasts togenerate iSNs. As exemplified herein, these include both embryonicfibroblasts and adult fibroblasts. The fibroblasts can be obtained orderived from various animal (e.g., mammal) species, e.g., human, mouseand rat. In some embodiments, stem cells can be used for conversion intoiSNs. Stem cells suitable for practicing the invention include and arenot limited to hematopoietic stem cells (HSC), embryonic stem cells,mesenchymal stem cells, and also induced pluripotent stem cells (iPSCs).Still some embodiments of the invention can utilize somatic cells otherthan fibroblasts such as blood cells. In these embodiments, blood cellsobtained from various organs including, e.g., liver, spleen, bone marrowand the lymphatic system, may all be employed in the practice of theinvention. In addition, methods of the invention may also be used forgenerating iSNs from peripheral blood cells such as erythrocytes,leukocytes and thrombocytes. In some other embodiments, the employednon-neuronal somatic cells can be glial cells (glia). Glia or glialcells refer to non-neuronal cells found in close contact with neurons,and encompass a number of different cells, including but not limited tothe microglia, macroglia, neuroglia, astrocytes, astroglia,oligodendrocytes, ependymal cells, radial glia, Schwann cells, satellitecells, and enteric glial cells.

V. Expressing Brn3a and Ngn1/Ngn2 in Non-Neuronal Cells

Co-expressing Brn3a/Ngn1 (BN1) genes, Brn3a/Ngn2 (BN2) genes, or theirfunctional variants described above in a non-neuronal cell can becarried out in accordance with the methods exemplified herein and othermethods well known in the art. Preferably, the genes are transientlyexpressed in the non-neuronal cell. This can be accomplished via cloningthe genes (genomic or cDNA sequences) into expression vector(s) and thenintroducing the expression vector(s) into the target non-neuronal cells.The two genes can be co-expressed from the same vector, as exemplifiedherein. Alternatively, the two genes can be cloned into and expressedseparately from two expression vectors. Preferably, the genes are clonedinto retroviruses or retroviral vectors (lentiviral vectors) fortransducing into the non-neuronal cells. As demonstrated in the Examplesbelow, a recombinant retroviral vector expressing the BN1 genes or theBN2 genes can be readily constructed by inserting the genes operablyinto the vector, replicating the vector in an appropriate packaging cellas described herein, obtaining viral particles produced therefrom, andthen infecting target non-neuronal cells (e.g., fibroblasts) with therecombinant viruses.

Cloning the BN1 or BN2 genes into expression vectors and expressing thegenes in non-neuronal cells can be performed using the specificprotocols described herein and methods routinely practiced in the art,e.g., as described in Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, N.Y., (3^(rd) ed., 2000); and Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(ringbou ed., 2003). Detailed procedures for cloning genes of interestinto lentiviral vectors, producing lentiviral viruses in packaging cells(e.g., 293T cells), and infecting host cells with the viruses forexpression of the genes are also described in the art, e.g., Boland etal. Nature 461, 91-94, 2009. Unless otherwise stated, other proceduresor steps required for practicing the present invention can be based onstandard procedures as described, e.g., in Murray et al., Gene Transferand Expression Protocols, The Humana Press Inc. (1991); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987); Current Protocols in ProteinScience (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: AManual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), Animal Cell Culture Methods (Methods in Cell Biology,Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1stedition, 1998).

In some embodiments, expression of the BN1 or BN2 genes in thenon-neuronal cells is controlled temporally. Temporal expression ofthese genes can be achieved via, e.g., the use of an inducibleexpression system. Any inducible expression method can be employed inthe practice of the present invention. For example, the expressionvectors can incorporate an inducible promoter that is active underenvironmental or developmental regulation, e.g., doxycycline(dox)-inducible lentiviral vectors. As exemplified herein, the genes canbe expressed under the control of a promoter that is activated whenbound by a reverse tetracycline transactivator (rtTA) and contacted bydoxycycline, tetracycline, or a tetracycline analog. The tTA protein iscreated by fusing one protein, TetR (tetracycline repressor), found inEscherichia coli bacteria, with the activation domain of anotherprotein, VP16, found in the Herpes Simplex Virus. The resulting tTAprotein is able to bind to DNA at specific TetO operator sequences. Inthe inducible expression system, several repeats of the TetO sequencesare placed upstream of a minimal promoter such as the CMV promoter. Theentirety of several TetO sequences with a minimal promoter is called atetracycline response element (TRE), because it responds to binding ofthe tetracycline transactivator protein tTA by increased expression ofthe gene or genes downstream of its promoter. Typically, in addition tothe expression vector(s) under the control of TetO for expressing theintended exogenous genes (e.g., the BN1 or BN2 genes), another vectorfor expressing a reverse tet transactivator is included in the inducibleexpression system.

The inducible expression system allows optimization of expression of theBN1 or BN2 genes in the non-neuronal cells that are appropriate theneurons to mature. The inducible expression system may also allow forhigher and/or more prolonged expression of the genes compared tonon-inducible expression systems. In some preferred embodiments of theinvention, induction of the BN1 or BN2 gene expression can last for atleast about 2 days, 4 days, 8 days, 12 days, e.g., between 2-8, 4-10,6-12, 8-14, 10-20, 12-30, or 15-40 days. The period of induction refersto the period from initial expression of the BN1 or BN2 genes (orinduction of expression with addition of doxycycline as exemplifiedherein) to the time the iSNs are selected (or termination of inductionwith doxycycline). Following introduction of the expression vectorsunder the control of TetO, expression of the BN1 or BN2 genes can beinduced in the non0neuronal cells with tetracycline, doxycycline, oranother tetracycline analog, and the cells can be cultured and selectedfor iSNs. Detailed protocols for inducible expression of exogenous genesin a host cell, e.g., using the rtTA/TetO system exemplified herein, arewell known in the art (e.g., WO 2011005580).

Retroviruses are a group of single-stranded RNA viruses characterized byan ability to convert their RNA to double-stranded DNA in infected cellsby a process of reverse-transcription. The resulting DNA then stablyintegrates into cellular chromosomes as a provirus and directs synthesisof viral proteins. The integration results in the retention of the viralgene sequences in the recipient cell and its descendants. The retroviralgenome contains three genes, gag, pol, and env that code for capsidproteins, polymerase enzyme, and envelope components, respectively. Asequence found upstream from the gag gene contains a signal forpackaging of the genome into virions. Two long terminal repeat (LTR)sequences are present at the 5′ and 3′ ends of the viral genome. Theseelements contain strong promoter and enhancer sequences and are alsorequired for integration in the host cell genome.

Retroviral vectors or recombinant retroviruses are widely employed ingene transfer in various therapeutic or industrial applications. Forexample, gene therapy procedures have been used to correct acquired andinherited genetic defects, and to treat cancer or viral infection in anumber of contexts. The ability to express artificial genes in humansfacilitates the prevention and/or cure of many important human diseases,including many diseases which are not amenable to treatment by othertherapies. For a review of gene therapy procedures, see Anderson,Science 256:808-813, 1992; Nabel & Felgner, TIBTECH 11:211-217, 1993;Mitani & Caskey, TIBTECH 11:162-166, 1993; Mulligan, Science 926-932,1993; Dillon, TIBTECH 11:167-175, 1993; Miller, Nature 357:455-460,1992; Van Brunt, Biotechnology 6:1149-1154, 1998; Vigne, RestorativeNeurology and Neuroscience 8:35-36, 1995; Kremer & Perricaudet, BritishMedical Bulletin 51:31-44, 1995; Haddada et al., in Current Topics inMicrobiology and Immunology (Doerfler & Bohm eds., 1995); and Yu et al.,Gene Therapy 1:13-26, 1994.

To construct retroviral vectors for transient expression of the BN1 orBN2 genes, a polynucleotide encoding one or both of the genes isinserted into the viral genome in the place of certain viral sequencesto produce a viral construct that is replication-defective. In order toproduce virions, a producer host cell or packaging cell line isemployed. The host cell usually expresses the gag, pol, and env genesbut without the LTR and packaging components. When the recombinant viralvector containing the gene of interest together with the retroviral LTRand packaging sequences is introduced into this cell line (e.g., bycalcium phosphate precipitation), the packaging sequences allow the RNAtranscript of the recombinant vector to be packaged into viralparticles, which are then secreted into the culture media. The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for transducing host cells (e.g., fibroblasts orstem cells) in gene transfer applications.

Suitable host or producer cells for producing recombinant retrovirusesor retroviral vectors according to the invention are well known in theart (e.g., 293T cells exemplified herein). Many retroviruses havealready been split into replication defective genomes and packagingcomponents. For other retroviruses, vectors and corresponding packagingcell lines can be generated with methods routinely practiced in the art.The producer cell typically encodes the viral components not encoded bythe vector genome such as the gag, pol and env proteins. The gag, poland env genes may be introduced into the producer cell and stablyintegrated into the cell genome to create a packaging cell line. Theretroviral vector genome is then introduced-into the packaging cell lineby transfection or transduction to create a stable cell line that hasall of the DNA sequences required to produce a retroviral vectorparticle. Another approach is to introduce the different DNA sequencesthat are required to produce a retroviral vector particle, e.g. the envcoding sequence, the gag-pol coding sequence and the defectiveretroviral genome into the cell simultaneously by transient tripletransfection. Alternatively, both the structural components and thevector genome can all be encoded by DNA stably integrated into a hostcell genome.

The methods of the invention can be practiced with various retroviralvectors and packaging cell lines well known in the art. Widely usedretroviral vectors include those based upon murine leukemia virus(MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus(SIV), human immunodeficiency virus (HIV), and combinations thereof(see, e.g., Buchscher et al., J. Virol. 66:2731-2739, 1992; Johann etal., J. Virol. 66:1635-1640, 1992; Sommerfelt et al., Virol. 176:58-59,1990; Wilson et al., J. Virol. 63:2374-2378, 1989; Miller et al., J.Virol. 65:2220-2224, 1991; and PCT/US94/05700). Particularly suitablefor the present invention are lentiviral vectors. Lentiviral vectors areretroviral vector that are able to transducer or infect non-dividingcells and typically produce high viral titers. Lentiviral vectors havebeen employed in gene therapy for a number of diseases. For example,hematopoietic gene therapies using lentiviral vectors or gammaretroviral vectors have been used for x-linked adrenoleukodystrophy andbeta thalassaemia. See, e.g., Kohn et al., Clin. Immunol. 135:247-54,2010; Cartier et al., Methods Enzymol. 507:187-198, 2012; andCavazzana-Calvo et al., Nature 467:318-322, 2010. Methods of theinvention can be readily applied in gene therapy or gene transfer withsuch vectors. In some other embodiments, other retroviral vectors can beused in the practice of the methods of the invention. These include,e.g., vectors based on human foamy virus (HFV) or other viruses in theSpumavirus genera.

In particular, a number of viral vector approaches are currentlyavailable for gene transfer in clinical trials, with retroviral vectorsby far the most frequently used system. All of these viral vectorsutilize approaches that involve complementation of defective vectors bygenes inserted into helper cell lines to generate the transducing agent.pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., Proc. Natl. Acad. Sci.U.S.A. 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeuticvector used in a gene therapy trial (Blaese et al., Science 270:475-480,1995). Transduction efficiencies of 50% or greater have been observedfor MFG-S packaged vectors (Ellem et al., Immunol Immunother. 44:10-20,1997; Dranoff et al., Hum. Gene Ther. 1:111-2, 1997). Many producer cellline or packaging cell line for transfecting retroviral vectors andproducing viral particles are also known in the art. The producer cellto be used in the invention needs not to be derived from the samespecies as that of the target cell (e.g., human target cell). Instead,producer or packaging cell lines suitable for the present inventioninclude cell lines derived from human (e.g., HEK 293 cell or 293T cell),monkey (e.g., COS-1 cell), mouse (e.g., NIH 3T3 cell) or other species(e.g., canine). Some of the cell lines are disclosed in the Examplesbelow. Additional examples of retroviral vectors and compatiblepackaging cell lines for producing recombinant retroviruses in genetransfers are reported in, e.g., Markowitz et al., Virol. 167:400-6,1988; Meyers et al., Arch. Virol. 119:257-64, 1991 (for spleen necrosisvirus (SNV)-based vectors such as vSNO21); Davis et al., Hum. Gene.Ther. 8:1459-67, 1997 (the “293-SPA” cell line); Povey et al., Blood92:4080-9, 1998 (the “1MI-SCF” cell line); Bauer et al., Biol. BloodMarrow Transplant. 4:119-27, 1998 (canine packaging cell line “DA”);Gerin et al., Hum. Gene Ther. 10:1965-74, 1999; Sehgal et al., GeneTher. 6:1084-91, 1999; Gerin et al., Biotechnol. Prog. 15:941-8, 1999;McTaggart et al., Biotechnol. Prog. 16:859-65, 2000; Reeves et al., Hum.Gene. Ther. 11:2093-103, 2000; Chan et al., Gene Ther. 8:697-703, 2001;Thaler et al., Mol. Ther. 4:273-9, 2001; Martinet et al., Eur. J. Surg.Oncol. 29:351-7, 2003; and Lemoine et al., I. Gene Med. 6:374-86, 2004.Any of these and other retroviral vectors and packaing producer celllines can be used in the practice of the present invention.

Many of the retroviral vectors and packing cell lines used for genetransfer in the art can be obtained commercially. For example, a numberof retroviral vectors and compatible packing cell lines are availablefrom Clontech (Mountain View, Calif.). Examples of lentiviral basedvectors include, e.g., pLVX-Puro, pLVX-IRES-Neo, pLVX-IRES-Hyg, andpLVX-IRES-Puro. Corresponding packaging cell lines are also available,e.g., Lenti-X 293T cell line. In addition to lentiviral based vectorsand packaging system, other retroviral based vectors and packagingsystems are also commercially available. These include MMLV basedvectors pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell linessuch as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 andAmphoPack 293, as well as NIH/3T3-based packaging cell line RetroPackPT67. Any of these and other retroviral vectors and producer cell linesmay be employed in the practice of the present invention.

VI. Therapeutic and Other Applications

Induced sensory neurons (iSNs) produced in accordance with the presentinvention can find various therapeutic and non-therapeutic applications.They can be used in cell replacement therapy to treat, amelioratesymptoms of, or prevent development of various diseases, e.g.,neurological conditions or disorders that are associated with ormediated by a loss or degeneration of sensory neurons, as well asdisorders that are associated with or caused by aberrantly functioningsensory neurons. In these applications, iSN cells prepared fromnon-neuronal cells of the subject in need of treatment can betransplanted or transferred to the same subject suffering from any of awide range of diseases or disorders associated with or mediated bysensory neuron death or degeneration. The transplanted cells canreconstitute or supplement differentiating or differentiated sensoryneurons in the subject. These therapeutic applications may be directedeither to treating the cause of the disease or to treating the effectsof the disease or condition. For example, therapy may be directed atreplacing sensory neurons whose death or degeneration caused thedisease, e.g., various sensory neuronopathies. The therapies can also beused in replacing sensory neurons that died as a result of the disease,e.g., ocular disorders such as age related macular degeneration (AMD).

Some embodiments of the invention are intended for treating sensoryneuronopathies or sensory neuron diseases. Sensory neuronopathiesencompass of a group of paraneoplastic, dysimmune, toxic, or idiopathicdisorders or conditions that are characterized by degeneration ofperipheral sensory neurons in dorsal root ganglia. Examples includeparaneoplastic sensory neuronopathy, hereditary sensory and autonomicneuropathy, HIV infection, Sjogren's syndrome, various connectivediseases, Freidreich's ataxia, and other rare idiopathic cases. See,e.g., Hainfellner et al., Ann. Neurol., 39:543-7, 1996; Kurokawa et al.,J. Neurol. Neurosurg. Psychiatry 65: 278-9, 1998; Lodi et al., Antioxid.Redox Signal. 8: 438-43, 2006; and Colli et al., Surg Neurol., 69:266-73,2008. Sensory neuronopathies are frequently associated withlife-threatening diseases such as cancer or potentially treatablediseases such as immune-mediated diseases. Sensory neuron diseasessuitable for treatment with methods of the invention include sensoryneuronopathies associated with immune-mediated and neoplastic diseases,and viral infections, and vitamin intoxication, and neurotoxic drugs.See, e.g., Horwich et al., Ann Neurol, 2: 7-19, 1977; Griffin et al.,Ann Neurol, 27: 304-315, 1990; Merchut et al., Neurol., 43: 2410-2411,1993; Scaravilli et al., Acta. Neuropathol. (Berl), 84: 163-170, 1992;Rubin et al., Muscle Nerve, 22: 1607-1610, 1999; Ramos et al., RevNeurol, 28: 1067-1069, 1999; Shimazaki et al., J. Neurol Sci, 194:55-58, 2002; Schaumburg et al., N Engl. J. Med, 309: 445-448, 1983; andQuasthoff et al., J. Neurol., 249: 9-17, 2002.

Some other embodiments of the invention are directed to treating ocularneovascularization or vascular degenerative disorders that areassociated with or caused by problems and decay of vision relatedsensory neurons. The five basic classes of neurons within the retina arephotoreceptor cells, bipolar cells, ganglion cells, horizontal cells,and amacrine cells. Examples of diseases or conditions associated withthese sensory neurons include macular degeneration, degeneration of thecentral visual field due to either cellular debris or blood vesselsaccumulating between the retina and the choroid, thereby disturbingand/or destroying the complex interplay of neurons that are presentthere. Other examples include glaucoma, loss of retinal ganglion cellswhich causes some loss of vision to blindness, and diabetic retinopathy,which is related to poor blood sugar control due to diabetic damages ofthe tiny blood vessels in the retina. Additional ocular vasculardisorder that may be treated with methods of the invention includeischemic retinopathy, iris neovascularization, intraocularneovascularization, corneal neovascularization, retinalneovascularization, choroidal neovascularization, diabetic retinalischemia, and retinal degeneration.

The subjects suitable for treatment with methods of the invention can beneonatal, juvenile or fully mature adults. In some embodiments, thesubjects to be treated are neonatal subjects suffering from a disease ordisorder noted above. In some preferred embodiments, the subjects arehuman, and the iSN to be used in the treatment are human cells,preferably autologous cells isolated from the same subject to betreated. In the various therapeutic applications of the invention, aniSN population is typically first generated with non-neuronal cells(e.g., fibroblasts or glial cells) from the subject in need oftreatment, using methods described herein. The iSN population can thenbe transferred to, or close to, an injured site in the subject.Alternatively, the cells can be introduced to the subject in a mannerallowing the cells to migrate, or home, to the injured site. Thetransferred cells may advantageously replace the damaged or injuredcells and allow improvement in the overall condition of the subject. Insome instances, the transferred cells may stimulate tissue regenerationor repair. In some embodiments, the induced sensory neurons may betransplanted directly to an injured site to treat a sensory neuronopathyor neurological condition. The iSN replacement therapies can beperformed with protocols well known in the art for cell transplantation.See, e.g., Morizane et al., Cell Tissue Res., 331(1):323-326, 2008;Coutts and Keirstead, Exp. Neurol., 209(2):368-377, 2008; and Goswamiand Rao, Drugs, 10(10):713-719, 2007. Other techniques and specificprocedures for carrying out the therapeutic methods of the invention canbe based on or modified from methods well known in the art. See, e.g.,Areman et al., Cellular Therapy: Principles, Methods, and Regulations,American Association of Blood Banks (AABB), 1^(st) ed., 2009; Wingard etal., Hematopoietic Stem Cell Transplantation: A Handbook for Clinicians,American Association of Blood Banks (AABB); 1^(st) ed., 2009; andRemington: The Science and Practice of Pharmacy, Mack Publishing Co.,20^(th) ed., 2000.

In general, the number of iSN cells to be administered to the subjectshould be sufficient for arresting the disease state, e.g., at leastabout 1×10², at least about 1×10³, at least about 1×10⁴, at least 1×10⁵,or at least 1×10⁶ cells. The number of cells to be administered maydepend upon the severity of the disease or condition, the age of thesubject and other factors that will be readily apparent to one ofordinary skill in the art. The cells may be administered in a singledose or by multiple dose administration over a period of time, as may bedetermined by the physician in charge of the treatment. Also, the numberof cells and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, a relatively low number of cells may be administered atrelatively infrequent intervals over a long period of time. Somesubjects may continue to receive treatment for the rest of their lives.In therapeutic applications, a relatively high number of cells atrelatively short intervals may be required until progression of thedisease is reduced or terminated, and preferably until the subject showspartial or complete amelioration of symptoms of the disease or disorder.Thereafter, the subject can be administered a prophylactic regime.

Other than therapeutic applications, iSNs generated with methods of theinvention can be used as a basic research or drug discovery tool. Someembodiments of the invention are directed to identifying agents ormodulations that can promote formation of iSNs from non-neuronal cells,as detailed below. Some other embodiments of the invention are directedto identifying compounds that can relieve pain and itch, or compoundsthat have unwanted off target effects causing pain and itch. In theseembodiments, iSNs are first generated in accordance with the presentinvention. The cells are then contacted with candidate agents toidentify compounds capable of modulating (e.g., inhibiting or enhancing)nociception/puritoception of the neurons. Still some other embodimentsof the invention relate to identifying compounds that can relieve orcause degeneration of various sensory neurons types. Similarly, thesemethods entail first generating a specific sensory neuron type inaccordance with methods of the invention. The induced neurons are thencontacted with candidate agents to detect one or more compounds that areable to modulate (promote or suppress) the survival or function of theneurons.

Some other embodiments are directed to evaluating the phenotype of agenetic disease, e.g., to better understand the etiology of the disease,to identify target proteins for therapeutic treatment, to identifycandidate agents with disease-modifying activity. These methods allowidentification of compounds with desired therapeutic activities, e.g.,an activity in modulating the survival or function of sensory neurons ina subject suffering from a neurological disease or disorder, e.g., toidentify an agent that will be efficacious in treating the subject. Forexample, a candidate agent may be added to a cell culture comprisingiSNs derived from the subject's somatic cells, and the effect of thecandidate agent assessed by monitoring output parameters such as iSNsurvival, the ability of the iSNs to become depolarized, the extent towhich the iSNs form synapses, and the like, by methods described hereinand in the art. Detailed procedures for the various screening methods ofthe invention can be based on or modified from methods well known in theart, or the exemplified methods herein for identifying agents thatpromote iSN formation.

In the various applications of the iSNs of the invention, at least oneparameter is monitored. The monitored parameter is a quantifiablecomponent of cells, particularly a component that can be accuratelymeasured, desirably in a high throughput system. The parameter can beany cell component or cell product including cell surface determinant,receptor, protein or conformational or posttranslational modificationthereof, lipid, carbohydrate, organic or inorganic molecule, nucleicacid, e.g., mRNA, DNA, etc. or a portion derived from such a cellcomponent or combinations thereof. While most parameters will provide aquantitative readout, in some instances a semi-quantitative orqualitative result will be acceptable. Readouts may include a singledetermined value, or may include mean, median value or the variance, andetc. Characteristically a range of parameter readout values will beobtained for each parameter from a multiplicity of the same assays.Variability is expected and a range of values for each of the set oftest parameters will be obtained using standard statistical methods witha common statistical method used to provide single values.

In some related embodiments, the invention provides kits orpharmaceutical combinations for generating iSNs and for using the iSNsin various applications described herein. Some of the kits will containone or more components of the agents described herein for inducingformation of sensory neurons from non-neuronal cells. Any of thecomponents described above may be provided in the kits, e.g., thespecific BN1 or BN2 encoding polynucleotides or expressing vectorsharboring them, packaging cell lines for producing recombinant viruses,as well as reagents for transducing recombinant viruses into anon-neuronal cell. The kits may further include non-neuronal cells forconversion into iSNs. The kits may also include tubes, buffers, etc.,and instructions for use. The various components of the kits may bepresent in separate containers, or some or all of them may bepre-combined into a reagent mixture in a single container, as desired.In addition to the above components, the subject kits may furtherinclude instructions for practicing the methods of the invention. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another form of these instructions is a computerreadable medium, e.g., diskette, compact disk (CD), etc., on which theinformation has been recorded. Yet another form of these instructionsthat may be present is a website address which may be used via theinternet to access the information.

VII. Screening for Factors that Stimulate Formation of iSNs fromNon-Neuronal Cells

Utilizing the system for generating iSNs described herein, the inventionalso provides methods to screen for compounds, cellular factors ormodulations that can promote or stimulate conversion of a non-neuronalcell into iSN. The compounds or cellular factors or manipulations can beexogenous compounds and genetic or epigenetic modulations inside thenon-neuronal cell. In these methods, co-expressing BN1 or BN2 in thenon-neuronal cell is performed in the presence of the candidatecompounds or cellular factors. This allows identification of specificcandidate factor (e.g., a miRNA or an epigenetic modulation) which canenhance the efficiency of conversion of the non-neuronal cell into iSN.Various biochemical and molecular biology techniques or assays wellknown in the art can be employed to practice the screening methods ofthe present invention. Such techniques are described in, e.g., Handbookof Drug Screening, Seethala et al. (eds.), Marcel Dekker (1P^(stP) ed.,2001); High Throughput Screening: Methods and Protocols (Methods inMolecular Biology, 190), Janzen (ed.), Humana Press (1P^(stP) ed.,2002); Current Protocols in Immunology, Coligan et al. (Ed.), John Wiley& Sons Inc (2002); Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press (3P^(rdP) ed., 2001); and Brent et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringboued., 2003).

The candidate compounds that can be screened for promoting iSN formationcan be any polypeptides, beta-turn mimetics, polysaccharides,phospholipids, hormones, prostaglandins, steroids, aromatic compounds,heterocyclic compounds, benzodiazepines, oligomeric N-substitutedglycines, oligocarbamates, polynucleotides (e.g., miRNAs or siRNAs),polypeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Some candidatecompounds are synthetic molecules, and others natural molecules.

By way of example, the screening methods of the present inventiontypically involve inducing sensory neuron formation as described hereinin the presence of candidate compounds or cellular manipulations (e.g.,epigenetic modulations). Thus, co-expression of BN1 or BN2 genes in thenon-neuronal cell (e.g., a fibroblast) is performed in the presence ofthe candidate compounds (e.g., miRNA) or performed in combination withother modulations (e.g., alternations in DNA methylation). If thepresence of a candidate agent or modulation leads to an enhancedconversion efficiency of the non-neuronal cells into iSNs, the candidatecompound (or the specific modulation) is then identified as an agent orfactor that promotes formation of iSNs. An enhanced conversionefficiency refers to any substantial increase in the number of theinitial non-neuronal cells being converted into iSNs. This can be anincrease of at least 20%, at least 30%, at least 40%, at least 50%, atleast 75%, or at least 90% or more, of the cells being converted intoiSNs.

In some methods, various transcription factors or other polypeptides arescreened for ability to promote iSN formation. Screen for iSN-promotingtranscription factors or other DNA-binding proteins can be performed byusing and/or modifying various assays that have been described in theart. See, e.g., Wiese et al., Front. Neurosci., 6:1-15, 2012; Alvaradoet al., J. Neurosci., 31(12):4535-43, 2011; Ouwerkerk et al., MethodsMol. Biol., 678:211-27, 2011; Laurenti et al., Nat. Immunol. 14,756-763, 2013; and Xu et al., Virol. 446:17-24, 2013. Some other methodsof the invention are directed to identifying nucleic acid agents thatare capable of stimulating formation of iSNs. For example, candidatemiRNAs can be co-expressed inside the non-neuronal cell. ExpressingmiRNAs in a host cell and testing the miRNAs for ability to enhance iSNformation can be performed with techniques based on or derived from anumber of miRNA screens that have been described in the literature. See,e.g., Voorhoeve et al., Cell, 124: 1169-1181, 2006; Becker et al., PLoSONE 7(11): e48474, 2012; Lam et al., Mol. Cancer Ther. 9: 2943-2950,2010; and Olarerin-George et al. BMC Biol., 11:19, 2013.

In still some methods, the candidate compounds are small organicmolecules (e.g., molecules with a molecular weight of not more thanabout 500 or 1,000). Preferably, high throughput assays are adapted andused to screen for such small molecules. In some methods, combinatoriallibraries of small molecule test agents can be readily employed toscreen for small molecule modulators that enhance iSN formation. Anumber of assays known in the art can be readily modified or adapted inthe practice of these screening methods of the present invention, e.g.,as described in Schultz et al., Bioorg Med Chem Lett 8: 2409-2414, 1998;Weller et al., Mol Divers. 3: 61-70, 1997; Fernandes et al., Curr OpinChem Biol 2: 597-603, 1998; and Sittampalam et al., Curr Opin Chem Biol1: 384-91, 1997.

EXAMPLES

The following examples are offered to further illustrate, but not tolimit the present invention.

Example 1 Some Experimental Techniques Used for Generating iSNs

Embryonic fibroblasts isolation and derivation: Wild-type CD1 mice werebred at the TSRI animal facility. MEFs were isolated from E14.5 embryosunder a dissection microscope. The head, internal organs and spinalcolumn containing the dorsal root ganglion was removed and discarded toeliminate cells with neurogenic potential. The remaining tissue wasmanually disassociated in 0.25% trypsin (Gibco) for 10 minutes at 37° C.subsequently the digestion solution was diluted and removed viacentrifugation. The resulting cells were seeded at approximately 3×10⁶cells/cm². MEFs were grown to confluency and passaged at least twiceprior to use. For HEF differentiation, human iPSCs colonies wereharvested using 1 mg/ml collagenase type IV and differentiated byEmbryoid Bodies formation. The EBs were cultured for 7 days innon-adherent suspension culture dishes (Corning), 2 days in 20% KSRmedium and the following 5 days in 10% FBS DMEM. On day 8 the EBs wereplated onto adherent tissue culture dishes and passaged according toprimary fibroblast protocols using trypsin for three passages before thestart of experiments.

Molecular cloning, cell culture and lentiviral infection: The cDNAs forhuman BRN3A (97% homologous to mouse Brn3a peptide), and mouse Ngn1, andNgn2 were cloned into lentiviral constructs under the control oftetracycline operator (TetO) using the following primers: BRN3A forwardand reverse respectively, 5′-ATGATGTCCATGAACAGCAAGCAG (SEQ ID NO:1) and5′-TCAGTAAGTGGCAGAGAATTTC (SEQ ID NO:2). Replication-incompetentVSVg-coated lentiviral particles were packaged in 293T cells asdescribed in Boland et al. Nature 461, 91-94, 2009. Passage three CD1MEFs were infected with lentivirus in MEF media (DMEM+10% fetal bovineserum and penicillin/streptomycin). After 12-16 hours of infectionmedia-containing virus was replaced with fresh MEF media. Transcriptionfactors were induced 48 hours post infection media by switching to MEFmedia supplemented with 5 μM doxycycline (Sigma). Four days afterinitiating induction with doxycycline media was replaced with N3 media(Vierbuchen et al., Nature 463, 1035-1041, 2010). Seven days postinduction doxycycline was withdrawn unless otherwise stated. Ten dayspost-induction was switched to neural maintenance media, which consistedof a 1:1 mix of DMEM/F12 (Invitrogen) and Neurobasal supplemented withB27, and NGF, BDNF and GDNF, all at 10 ng/ml. Efficiency of conversionwas measured by the number of Map2-positive cells divided by to theinitial number of cells plated.

Immunohistochemistry, and RT-PCR: For immunofluorescence staining, cellswere fixed 4% paraformaldehyde for 10 minutes at room temperature. Cellswere then washed three times with PBS and subsequently permeabilizedwith 0.1% Triton X-100 (Sigma) in PBS. After washing andpermeabilization cells were blocked in 5% horse serum for thirty minutesat room temperature. Primary staining was performed overnight at 4° C.in block. Secondary antibodies were diluted in blocking solution andstained room temperature for one hour. EdU staining was performed usingClick-it EdU kit (C10337; Invitrogen) following manufacturer'sinstructions. The following antibodies and dilutions were used:Ms-βIII-Tubulin (Tuj1) (1:1000, Covance MMS-435P); Rb-βIII-Tubulin(Tuj1) (1:1000, Covance MRB-435P); Ms-Map2 (1:500, BD 556320); Ms-VGlut1(1:100, Millipore MAB5502); Rb-Vglut2 (1:50, abcam ab72310); Gp-VGlut3(1:1000, Millipore AB5421); Ms-Brn3a (1:200, Millipore MAB1585);Gt-human Ret (1:100, R&D AF1485); Gt-mouse Ret (1:100, R&D AF482);Ms-Islet1 (1:200, DSHB 40.2D6); Gt-mouse TrkB (1:200, R&D BAF1494);Gt-TrkA (1:200, R&D AF175); Sh-TrkC (1:200, Abcam ab72120); Ms-NF200(1:200, Millipore MAB5266); Rb-Runx1 (1:100, Novus Bio NBP1-61277);Rb-Peripherin (1:200, Millipore AB1530); Gaba (Sigma), Ngn1 (1:500Abcam), Ngn 2 (1:500Millipore), P75 (Abcam), CGRP (1:500 Neuromics),Substance P (1:500 Neuromics), VAMP (Synaptic systems), Synapsin(Synaptic systems). Secondaries: A21447D-G647 A10036-DM546 A21202-DM488A10040-DR546 A11015-DSh488 A11056-DGt546 A21206-DRb488 A21208-DRt488A21098-DSh546.

For RT-PCR analysis total RNA was isolated at the time-points indicatedusing Trizol (Invitrogen) following manufacturer's instructions, treatedwith DNaseI (Ambion) and 1.0 μg was reverse transcribed with iScript(BioRad). PCRs were performed using TaqMan Gene Expression Assays(Applied Biosystems) or SYBR green. For quantitative RT-PCR from singlecells, single cells were grown on glass coverslips from which they wereisolated three weeks after induction using a using a patch pipet andmicromanipulator. Cells were placed in 4 μl of lysis/RT bufferconsisting of Superscript III RT buffer (Invitrogen) supplemented with0.5% NP-40, 1 mM DTT, and SuperRnase inhibitor (Ambion), and Prime RNaseinhibitor (5 Prime). Cells were spun down in a microcentrifuge and flashfrozen at −80° C. until further processing. Reverse transcription wasperformed using SuperScript III (Invitrogen) with 130 nM of eachgene-specific 3′-primers. Reverse transcription products were thansubjected to 15-cycles of target specific pre-amplification using 15 nMof outside nested primers designed to produce amplicons of 300-400 bp. Aquantitative real-time PCR with was subsequently performed using SYBRselect (Applied Biosystems, 4472918) with internal primers designed togenerate amplicons approximately 100 base pairs. To ensure specificitytemplate titrations were performed and only primers that demonstratedlinear amplifications were used melt-curves were also obtained forsingle cells and controls to ensure specificity of products.

Calcium imaging and electrophysiology: Calcium imaging was performed onmouse and human iSNs 2 to 3 weeks post-induction using Map2::GCAMP5.Glentiviral reporter (Addis et al., PLoS ONE 6, e28719, 2011). Imagingwas performed in Tyrode's solution (145 mM NaCl, 2.5 mM KCl, 10 mMHepes, NaH₂PO₄, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM Glucose, and 0.4 mMascorbic acid) at a constant flow rate of 250 ml/hour. To monitorcalcium response, capsaicin, menthol, and mustard oil were addedsequentially in randomized orders to the flow chamber at a 10×concentration to deliver a final concentration of 10 μM capsaicin, 100μM menthol, and 100 μM mustard oil. Each tracing experiment wasbracketed by an initial and final pulse of 5 mM KCl to confirm neuralidentity and sustained functional ability. Only cells with neuralidentity and sustained functional ability were analyzed. Calciumresponses were determined by calculating the change in fluorescence overthe initial fluorescence (F−F₀)/F₀, where F=the fluorescence at a giventime point and F₀=the mean basal, unstimulated fluorescence of eachcell. A typical non-response area was selected for fluorescence bleednormalization and background subtraction.

Electrophysiology: Fibroblasts were plated, transduced and cultured onlaminin coated thermanox plastic coverslips (13 mm) as described in cellculture methods. Coverslips were placed in the recording chamber mountedon an Olympus IX 71 microscope. Spontaneous or evoked responses wererecorded at room temperature via whole-cell recording with a patchelectrode. Signals were amplified using an Axopatch200B andMultiClamp700B (Axon Instruments) and filtered at 2 KHz via a Bessellow-pass filter. Data were sampled and analyzed using pClamp10.1software in conjunction with a DigiData interface (Model 1322A, 1440AAxon Instruments). Patch pipettes were pulled from standard wall glassof 1.5 mm OD (Warner Instruments) and had input resistances of 5-11 M.In general, for recording voltage-gated currents and action potentials,patch electrodes were filled with the following solution (in mM): 140K-gluconate, 5 NaCl, 1 MgCl2, 10 EGTA, 10 HEPES, 10 EGTA; pH adjusted byKOH to 7.25, osmolarity measured at 290 mOsm. For isolation of Na⁺current from K⁺ current, cesium was substituted for potassium as themajor cation in the patch pipette-filling solution in order to suppressK⁺ currents. The composition of the intracellular solution used forrecording ligand-gated currents was as follows (in mM): 130 Cs-gluconate2 MgATP, 1 MgCl2; 10 EGTA; 10 HEPES; pH 7.25, osmolarity 300 mOsm. Foeevoked action potentials we started recordings in the current clamp modeat the resting membrane potential values and use artificialhyperpolarization by negative current injection with A level of 25 pA.The bath solution generally contained a Na+ saline based Hank's balancedsalt solution (pH=7.3). To monitor voltage-gated currents, after initialpre-hyperpolarization to −90 mV for 300 ms to relieve Na⁺ inactivation,we applied step potentials ranging from −60 to +30 mV for 100 ms.

Example 2 Generation of Sensory Neurons from Mouse Embryonic Fibroblast

To determine whether we could produce induced neurons using factorsinvolved in the development of the somatic sensory neural lineage, weused lentiviral vectors to express either Brn3a and Ngn1 (BN1) or Brn3aand Ngn2 (BN2) in mouse embryonic fibroblasts (MEFs). Reprogrammingfactor expression was controlled temporally by coinfecting with alentivirus encoding the doxycycline inducible factor rtTA and placingthe BN1/BN2 factors under control of the doxycycline inducible promoterTetO (FIGS. 8a and b ). After eight days of induction we removeddoxycycline and allowed cells to mature without further expression ofthe exogenous reprogramming factors. Both the BN1 and BN2 conditionsproduced numerous Tuj1 positive cells exhibiting neuronal morphology andthe majority of these cells also expressed Map2 (FIG. 1a-b and FIG. 8b). This conversion requires both factors. Expression of either of eitherNgn1 or Ngn2 alone induced Tuj1-positive cells, however, these cells didnot express Map2 and did not exhibit neuronal morphologies. No TuJ1 orMap2 positive cells were found in untreated MEFs or those exposed onlyto Brn3a (FIG. 8b ). Immunostaining further confirmed that >98% ofMap2/Tuj1 double positive cells induced by Brn3a/Ngn1 or Brn3a/Ngn2expressed synapsin and synaptobrevin (also called VAMP) consistent witha relatively mature neural state capable of forming functional synapses(FIG. 1c-e ).

To determine whether BN1 and BN2 neural cells exhibit membrane andelectrophysiological properties of neurons, we performed whole-cellpatch-clamp recordings. The average resting membrane potential of neuralcells induced by BN1 or BN2 was −42.01 mV (SEM=1.74, n=4) which issimilar to resting potentials reported for other induced neurons(Vierbuchen et al., Nature 463, 1035-1041, 2010). Depolarizing involtage clamp mode elicited fast inward currents followed by slowoutward currents consistent with opening of voltage-activated sodium andpotassium channels, respectively (n=5; FIG. 1f ). Of the five cells thatexhibited Na+/K+ currents, four were able to fire evoked actionpotentials and one of these cells also exhibited spontaneous firing.(FIGS. 1g and h ). Taken together, these data show that BN1 and BN2 aresufficient to reprogram fibroblasts to neuronal cells with synaptic andelectrophysiological properties consistent with a neuronal identity.

The neurons induced with BN1 or BN2 exhibit molecular hallmarks of thesomatic sensory neural lineage. To determine the extent to which neuronsinduced by BN1 or BN2 resemble endogenous somatic sensory neurons, weexamined expression profiles of neurofilaments neurotransmitters, andneuropeptides commonly used to characterize populations of theseneurons. Within the DRG, distinct populations of sensory neurons, thin(C-fiber) and thick (A-fiber), can be identified by their expression ofeither peripherin or NF200, respectively. Peripherin is a type-IIIneuron specific intermediate filament found primarily in the peripheralnervous system. Approximately 60% of DRG neurons express peripherin,which corresponds to small diameter unmyelinated neural populations.Similar to DRG neurons, 50-60% of induced neurons (BN1=64.17%±9.67;BN2=51.93%±9.12) expressed peripherin (FIGS. 2a and b ). The majority ofremaining non-peripherin positive neurons express the heavyneurofilament NF-200 (also called NF-H), corresponding to thickmyelinated fibers. Similar to endogenous DRGs, approximately 30% of iSNs(BN1=29.38±1.46; BN2=30.60±3.11) expressed NF200 (FIGS. 2a and c ).These data demonstrate that the BN1 and BN2 transcription factorcombinations both induce neurons to express neurofilaments typicallyfound in peripheral sensory neurons in similar proportions to thoseobserved in the DRG. Somatic sensory neurons are excitatoryglutamatergic neurons that express three vesicular glutamatetransporters 1, 2 and 3 (also known as vGlut1(Slc17a7), vGlut2(slc17a6),and vGlut3(slc17a8), respectively). Consistent with a somatic sensoryneural identity, the induced neurons expressed all three vesicularglutamate transporters but did not express GABA, demonstrating that thisconversion produces excitatory but not inhibitory neuronal subtypes(FIG. 2a ; and FIG. 9a ). In the DRG a subset of nociceptive neuronsalso express the neuropeptide CGRP. We observed CGRP expression inapproximately 10% of induced neural cells (FIG. 2a ; and FIG. 9a ).These data demonstrate that neurons induced with BN1 and BN2 expresscharacteristic neurofilaments, neurotransmitters and peptides found inperipheral sensory neurons.

Mature peripheral sensory neurons express the transcription factors Isl1and Brn3a but no longer express Ngn1 or Ngn2. During reprogramming witheither BN1 or BN2 both Isl1 and endogenous Brn3a expression are stronglyup regulated and this expression is maintained for at least 14 daysafter doxycycline withdrawal (FIG. 2d ). In contrast, Ngn1 and Ngn2 wereno longer detectable after exogenous induction was extinguished (FIG. 9b). Treatment with BN1 or BN2 did not induce the expression of variousmarkers of other neuronal subtypes such as Satb2, Ctip2, Brn2, Tbr2, andTbx21 (data not shown). Finally, single cell RT-PCR confirmed thatinduced neurons that have up regulated Map2 and Isl1 have alsodown-regulated the fibroblast specific genes Snai1 and Fsp1 (FIG. 2e ).These data further support the conclusion that BN1 and BN2 reprogramfibroblasts to assume and maintain a neural identity consistent withthat of endogenous somatic sensory neurons.

In vivo, selective expression of one of the three Trk receptors, TrkA,TrkB, and TrkC is critical for proper target innervation, survival, andexpression of downstream molecular programs that distinguish the threelineages of sensory neurons. Quantitative RT-PCR showed that mRNA forTrkA, TrkB and TrkC was present as early as 4 days post induction inboth the BN1 and BN2 conditions. Trk receptor mRNA levels reach aplateau after 7 days, which is maintained for at least 22 days, showingthat conversion has become independent of the inducing factors. (FIG. 2f). Immunostaining confirmed that TrkA, TrkB, and TrkC proteins arepresent in the soma and along the axons of neurons induced with eitherBN1 or BN2 at 16 days post induction (FIG. 2h ). Immunostaining for eachof the Trk receptors labels approximately 25-30% of the induced neurons(FIG. 2g ), suggesting that they may each comprise a distinctnon-overlapping subpopulation as is seen in vivo.

To establish whether the Trk-staining corresponded to one homogenouspopulation expressing all three receptors, or three unique populationseach expressing one of the three Trk-receptors; we simultaneouslyco-stained for TrkA, B and C and each pair-wise combination of the threereceptors. Each pair-wise combination of TrkA, B, and C stainedapproximately 60% of neurons induced by BN1 and BN2 (FIG. 9c ). Incontrast, simultaneous co-staining for all three Trk receptors labeledapproximately 90% of induced neurons (BN1=88.95%±2.17;BN2=91.09%±1.80)(FIG. 2g). In the DRG most of the sensory neurons alsoexpress the p75^(Ngfr) the low-affinity NGF receptor. In our experimentsthe majority (>80%) of total neurons induced by BN1 and BN2 expressp75^(Ngfr) (FIGS. 2g and i ). Together, these results suggest that TrkA,TrkB, and TrkC-positive cells represent three distinct populations thatconstitute the majority of neurons induced by BN1 and BN2. Becauseneurons induced by BN1 or BN2 express transcription factors,neurofilaments, neurotransmitters and neuropeptides, characteristic ofsomatic sensory neurons and selectively express one of the three majorTrk receptor lineage markers, we chose to designate this neuralpopulation as induced sensory neurons (iSNs) or induced somatic sensorylineage neurons (iSLNs). Intriguingly, although in vivo studies suggestthat Ngn1 and Ngn2 expression might bias differentiation to differentTrk receptor sublineages, treatment with BN1 and BN2 produced equalproportions of cells expressing each lineage marker across multipleexperiments (Ma et al., Genes Dev. 13:1717-28, 1999).

Example 3 Characterization of iSNs Generated from MEFs

We observed that the reprogramming described herein induces somaticsensory neural morphology. In vivo, sensory neurons also vary withrespect to the size of their cell body. TrkA-positive neurons are thesmallest, TrkB-positive neurons are intermediate, and TrkC-positiveneurons comprise a population with the largest cell bodies. To determinewhether iSNs generated with either BN1 or BN2 also adopt thismorphological signature we measured the area of TrkA, B, and C-positiveneurons. The induced neurons generated by both BN1 and BN2 exhibiteddistinct distributions of soma size that correlated with expression ofTrkA, TrkB, and TrkC (FIG. 3a , and FIGS. 10a-b ). The mean soma area ofTrkA-positive cells (BN1=231.3 μm±14.02; BN2=237.8 μm±21.87) wassignificantly smaller than TrkB-positive cells (BN1=456.2 μm±34.38;BN2=397.5 μm±30.69). Likewise, the mean soma area of TrkB-positive cellswas significantly smaller than TrkC-positive neurons (BN1=569.9μm±47.01; BN2=598.5 μm±53.29) (FIG. 3a , and FIG. 10a-b ). These resultsfurther suggest that the TrkA, B and C positive cells we observedrepresent three distinct populations.

Sensory neurons of the DRG also exhibit a unique pseudounipolarmorphology characterized by the absence of dendrites and presence of asingle bifurcating axon. Visual inspection of the neurons induced by BN1and BN2 suggested that they had adopted this morphology (FIG. 3b ).Therefore we quantified the number of multipolar, bipolar, unipolar, andpseudounipolar neurons generated from two independent experiments usingBN1 and BN2. Remarkably, the majority of induced neurons(BN1=91.99%±0.40; BN2=84.69%±1.357) exhibited a pseudounipolarmorphology (FIG. 3c and FIG. 10c ). The remaining neural cells wereeither unipolar without observable bifurcation (BN1=6.13%±0.414;BN2=11.89%±1.43), or bipolar (BN1=1.89%±0.02, BN2=3.4%±0.08) (FIG. 3c ).In contrast, as reported previously, neurons generated using Brn2, Ascl1, and Zic1 (BAZ) were primarily multipolar (Vierbuchen et al., Nature463, 1035-1041, 2010); no pseudounipolar neurons were observed usingthese factors (FIG. 3c ). This finding demonstrates that directreprogramming with only two transcription factors can induce a highlyspecific neurite morphology distinct from that produced by previousmethods, predicting that exogenous cues such as appropriate targets orglial derived factors are not required for this aspect of neuronalspecification.

We also found that the induced iSNs include cells that resembledifferent populations of nociceptors. The utility of iSNs formechanistic and screening studies depends on the ability of theseneurons to recapitulate functional properties of biomedically relevantneural subtypes. Assays for distinguishing functional properties ofsensory neurons are predominantly limited to the TrkA nociceptivesub-lineage because TrkC lineage proprioceptive function requires muscleinnervation and the mechanical responses of the TrkB lineage are similarto the inherent mechanical responses of fibroblasts. Therefore wefocused on establishing the functional responses of the TrkA lineage ofnociceptive neurons. TrkA positive neurons in the DRG and trigeminalnerve detect sensations such as pain and temperature in part byexpressing various combinations of members of the Transient receptorpotential (Trp) family of ion channel receptors. To determine whetheriSNs exhibit key functional properties of sensory neurons, we firstassessed expression of receptor ion channels TrpV1, TrpM8, and TrpA1,which mediate a broad spectrum of sensations such as heat, cold, andnoxious chemicals, respectively. Uninfected MEFs, and neurons inducedwith the proneural cocktail Brn2, Mash1, Zic1 (BAZ) failed to expressany of the Trp channels (FIGS. 4a and b ). However, iSNs induced witheither BN1 or BN2 robustly expressed TrpA1, TrpM8, and TrpV1 (FIGS. 4aand b ). In addition, BN1 or BN2 iSNs selectively expressed thevoltage-gated sodium channel Nav1.7 (also called Scn9a) known to play acritical role in the generation and conductance of action potentials innociceptive neurons (FIGS. 4a and b ). Collectively, these data predictthat some iSNs should be able to sense and respond to stimuli such asheat, cold and pungent natural compounds.

To determine whether iSNs can respond functionally to Trp channelligands, we evaluated calcium transients following exposure to allylisothiocyanate (mustard oil), menthol, and capsaicin at concentrationsknown to selectively activate TrpA1, TrpM8 and TrpV1, respectively.Following an initial depolarization using 2.5 mM KCl to identifyresponsive iSNs, cells were serially washed with menthol, mustard oil,and capsaicin in a randomized order. Both BN1 and BN2 iSNs exhibitedrapid transient calcium fluctuations in response to capsaicin (BN119.7%±3.0 and BN2 21.2%±3.5); menthol (BN1 15.4%±2.6 and BN2 17.4%±4.3);mustard oil (BN1 12.9%±3.8 and BN2 12.5%±3.4) (FIG. 4c-e ). Although themajority of ligand responsive iSNs were responsive to only one ligand, afew iSNs responded to two ligands sequentially: capsaicin and menthol(BN1 1.7%±1.7% and BN2 0.7%±0.7); capsaicin and mustard oil (BN12.3%±2.3 and BN2 4.1%±1.7); menthol and mustard oil (BN1 1.7%±1.7 andBN2 0.7%±0.7). No iSNs responded to all three sensory ligands. While therelative proportions of cell responsive to various combinations ofstimuli differs from those seen in vivo, these results demonstrate thatiSNs can diversify and express the primary receptors and signalingpathways required to selectively respond to biomedically-relevantsensory stimuli.

Example 4 Reprogramming Does Not Require Cell Division or EmbryonicPrecursors

We examined whether the observed reprogramming of fibroblasts to thesomatic sensory lineage requires cell division or specialized embryonicprecursors. The induction of BN1 or BN2 in fibroblasts results in theproduction of multiple related subtypes of neurons (either TrkA, TrkB orTrkC positive), which seem to arise in roughly equivalent numbers. Onepossible explanation for this would involve a multipotent proliferatingprecursor cell that differentiates into these three subtypes. To addressthis possibility we performed two experiments. First, MEFs werereprogrammed in the presence of the proliferation marker EdU(5-ethynyl-2′-deoxyuridine, a click-chemistry BrdU analog). Addition ofEdU to the culture media three days post induction labeled fewer than0.5% of the total Map2-positive iSNs after 14 days of reprogramming,suggesting that BN1 and BN2 neural conversion does not involveproliferation (FIG. 5a-c ). Second, to further block proliferation weapplied the mitotic inhibitor arabinofuranosyl cytidine (AraC) tocultures continuously from three days post induction and quantified thenumber of Map2-positive cells on day 14. Consistent with the absence ofa proliferative intermediate, mitotic inhibition from day 3 did notsignificantly alter the number of neurons that were produced duringreprogramming (FIG. 5d-f ). These data indicate that cell division isnot required for induction of iSNs and predict that the diversificationof neurons may involve a stochastic cell fate choice that operates in anon-dividing cell.

While the frequency of iSNs that arise using this protocol is relativelyhigh (˜1-10%) and varies depending on viral titer, it remains possiblethat this conversion requires a particular embryonic cell type thatcould be “pre-committed” to this lineage. To test this we derivedtail-tip fibroblasts (TTFs) from 5-day old pups. Staining for markers ofneural precursors showed that these cultures did not contain neuralprogenitor (data not shown). When TTFs were exposed to BN1 or BN2 foreight days, and cultured without doxycycline induction for an additional7 days, Map2/Tuj1-positive cells were observed. As previously observed,these cells exhibited pseudounipolar morphologies consistent withsensory neural identity (FIGS. 5g, h ). Furthermore, neural cellsinduced with both BN1 and BN2 expressed all three of the TrkA, B, and Creceptors (FIG. 5h ). These results demonstrate that the ability of BN1and BN2 to generate neurons is not restricted to embryonic tissues andthat iSNs can indeed be generated from adult fibroblasts.

Example 5 Generation of iSNs from Human Fibroblasts

We further sought to determine whether this method could be applied tohuman cells. Human embryonic fibroblasts (HEFs) were derived from iPSCsusing established methods (Son et al., Cell Stem Cell 9, 205-218, 2011).HEFs were transduced with inducible lentiviral vectors encoding eitherBN1 or BN2 and the doxycycline dependent activator rtTA as described inthe mouse reprogramming experiments. Transcription factor expression wasinduced for eight days, followed by at least seven days removal fromdoxycycline induction. Fifteen days after induction, we observedMAP2/TUJ1-positive cells with neuronal morphologies in the BN1 and BN2conditions, but not in control uninfected HEFs or in HEFs treated withsingle transcription factors (FIG. 6a-b ). Similar to mouse iSNs, humanBN1 and BN2 induced neurons exhibit mRNA and protein expression ofsomatic sensory lineage markers TrkA, B, and C (FIG. 6c -e, m).Immunostaining of iSNs shows that TRKA (BN1 33.6%±6.2, BN2 35.8%±7.6),TRKB (BN1 28.6%±5.6, BN2 29.0%±0.5), and TRKC (BN1 30.0%±7.6, BN232.3%±1.7) are each expressed in approximately one third of thepopulation (FIG. 6c -e, n). Additionally they showed nearly ubiquitousexpression of the transcription factor ISL1 (BN1=93.15%±4.14;BN2=90.5±5.14) (FIG. 6f, n ). These markers were absent in neuronsderived using the pan-neural cocktail Brn2, Ascl1 , and Zic1 (BAZ) (FIG.6n ). However, both BN1/2 and BAZ neurons were shown to be mostlyglutamatergic through immunostaining of vGLUT2 (FIGS. 6g, n ). Thissuggests that a sensory neural phenotype is selectively imparted tohuman fibroblasts by transient ectopic expression of Brn3a with eitherNgn1 or Ngn2.

We further characterized the BN1 and BN2 iSNs and observed strongexpression of low-binding NGF receptor p75 in nearly all iSNs (FIGS. 6kand 6o ), and expression of the GDNF receptor c-RET in a subset of iSNs(FIGS. 6j and 6o ), which was absent from the mouse iSNs for unknownreasons. Furthermore, neural populations of iSNs expressed thecharacteristic neurofilaments, peripherin and NF200 that are associatedwith thin and thick fiber neurons, respectively (FIGS. 6h, 6i, and 6o ).Additionally BN1 and 2 showed expression of vGLUT1 (FIGS. 6l and 6o )and a complete absence of GABA (data not shown). Lastly, we testedwhether reprogramming with BN1 and BN2 could convert neonatal humanfibroblasts to neurons. Twenty days of reprogramming led to MAP2/TUJ1positive cells with neuronal morphologies, that express ISL1 and lineagemarker TRKA and TRKB, demonstrating that induction of iSNs is notrestricted to embryonic fibroblasts (data not shown).

We additionally examined whether human iSNs exhibit membrane andelectrophysiological properties of mature neurons. We performedwhole-cell patch-clamp recordings. The average resting membranepotential of human iSNs was 40.067 mV (SEM=3.724, n=15). Depolarizing involtage clamp mode elicited fast inward currents followed by slowoutward currents consistent with opening of voltage-activated sodium andpotassium channels, respectively (FIG. 7a ). In addition, human iSNswere able to fire action potentials (FIG. 7b ). Collectively, these datademonstrate human iSNs possess membrane and electrophysiologicalproperties of functional neurons.

To determine whether human iSNs had acquired key characteristics ofvarious subpopulations of nociceptive neurons we assessed expression ofTRPA1, TRPM8, TRPV1 and NAV1.7 in HEFs, BAZ neurons and the iSNs. Noexpression of TRPA1, TRPM8, TRPV1 or NAV1.7 was detected in HEFs or BAZneurons. In contrast, robust expression was observed in the human iSNsgenerated with both BN1 and BN2 (FIG. 7c ). To determine if the TRPchannels were functional, we performed calcium-imaging studies aspreviously described for the mouse iSNs. Human iSNs generated witheither BN1 or BN2 exhibited rapid and selective calcium transients inresponse to capsaicin (BN1 4.4%±1.7 and BN2 5.9%±3.3); menthol (BN14.4%±1.4 and BN2 6.0%±2.3); mustard oil (BN1 12.8%±4.5 and BN28.8%±3.7). The majority of KCl responsive iSNs did not respond to any ofthe three ligands (BN1 83.1%±4.9 and BN2 82.6%±3.4) (FIGS. 7d and 7e ).A few iSNs responded to two ligands sequentially: capsaicin and mustardoil (BN1 3.2%±0.7 and BN2 1.2%±1.2); menthol and mustard oil (BN11.9%±1.9 and BN2 1.1%±1.1). We did not detect any neurons thatsimultaneously responded to both capsaicin and menthol, or all threeligands. These calcium fluxes in response to ligand specific activationsuggest that the channel receptors TRPA1, TRPM8, and TRPV1 are indeedfunctional though perhaps not co-expressed in similar numbers of cellsas would expected based on in vivo studies (FIG. 7d-f ).

In addition to nociceptive stimuli, subsets of somatic sensory neuronsrespond to various mediators of itch. Importantly, the pathwaysregulating human and mouse itch are not well defined and appear todiffer between species. Here we wished to determine whether subsets ofour human iSNs would respond to known puritogenic compounds such ashistamine, chloroquine, and the peptides BAM 8-22 and SLI-GLR, whichwould suggest that these cells could be used to further investigate themolecular mechanisms of itch sensation in humans. Indeed, subsets ofhuman iSNs robustly responded to histamine (BN2 28.1%±14.6) andchloroquine (BN2 44.1%±12.6), while BAM 8-22 (BN2 25.0%±25.0) andSLI-GLR (BN2 9.2%±3.3) elicited weaker responses in smaller subsets ofneurons (FIG. 7g-i ). Again we also observed neurons that responded tovarious pair wise combinations of puritogenic compounds, as has beenreported in mouse studies (FIG. 7i ). These results indicate that it maybe possible to further delineate the molecular basis of human itchsensation using iSNs since these cells are readily available andamenable to genetic manipulation.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. Although any methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods and materialsare described.

All publications, GenBank sequences, ATCC deposits, patents and patentapplications cited herein are hereby expressly incorporated by referencein their entirety and for all purposes as if each is individually sodenoted.

We claim:
 1. A method for generating induced sensory neurons (iSNs),comprising co-expressing Brn3A and Ngn2 genes via one or more expressionvectors harboring Brn3A and Ngn1 genomic or cDNA sequences in anon-neuronal cell, thereby generating induced sensory neurons, whereinthe non-neuronal cell is a fibroblast or a stem cell from a mammal. 2.The method of claim 1, wherein expression of the Brn3A/Ngn2 genes istemporal.
 3. The method of claim 2, wherein temporal expression of theBrn3A/Ngn2 genes is via inducible expression.
 4. The method of claim 1,wherein the non-neuronal cell is a fibroblast, an embryonic stem cell(ESC), or an induced pluripotent stem cell (iPSC).
 5. The method ofclaim 1, wherein the non-neuronal cell is an embryonic fibroblast or anadult fibroblast.
 6. The method of claim 1, wherein the mammal is human,mouse or rat.
 7. The method of claim 1, wherein an expression vectorharboring the Brn3A gene and the Ngn2 gene is introduced into thenon-neuronal cell.
 8. The method of claim 7, wherein the expressionvector is a lentiviral vector.
 9. The method of claim 1, furthercomprising detecting in the induced sensory neurons expression of one ormore neuronal markers.
 10. An induced sensory neuron generated inaccordance with claim
 1. 11. An isolated cell comprising one or moreexpression vectors, wherein the expression vectors express a combinationof Brn3A/Ngn1 genes or Brn3A/Ngn2 genes.
 12. A method for treating in asubject a neurological condition or disorder that is associated with ormediated by a loss or degeneration of sensory neurons, comprising (1)obtaining a non-neuronal cell from the subject in need of treatment; (2)generating a population of induced sensory neurons (iSNs) from saidnon-neuronal cell by co-expressing a combination of Brn3A/Ngn1 genes orBrn3A/Ngn2 genes via one or more expression vectors in the non-neuronalcell; and (3) administering a therapeutically effective amount of theiSN population to the subject, thereby treating the neurologicalcondition or disorder in the subject.
 13. The method of claim 12,wherein expression of the Brn3A/Ngn1 genes or Brn3A/Ngn2 genes istemporal.
 14. The method of claim 13, wherein temporal expression of theBrn3A/Ngn1 genes or Brn3A/Ngn2 genes is via inducible expression. 15.The method of claim 12, wherein the non-neuronal cell is a fibroblast,an embryonic stem cell (ESC), or an induced pluripotent stem cell(iPSC).
 16. The method of claim 12, wherein the non-neuronal cell is anembryonic fibroblast or an adult fibroblast.
 17. The method of claim 12,wherein an expression vector harboring the Brn3A/Ngn1 genes orBrn3A/Ngn2 genes is introduced into the non-neuronal cell.
 18. Themethod of claim 17, wherein the expression vector is a lentiviralvector.
 19. The method of claim 12, further comprising examining theinduced sensory neurons for the presence of a neuronal marker.
 20. Themethod of claim 12, wherein the subject is a human.